Method for determining the amount of a therapeutic antibody in the brain

ABSTRACT

Herein is reported a method for determining the concentration of a therapeutic antibody in a tissue of an experimental animal to whom the therapeutic antibody had been administered, wherein the interference from residual blood in a tissue sample of the experimental animal, which is used for determining the concentration of the therapeutic antibody in said tissue, is reduced, wherein the concentration of the therapeutic antibody in the tissue of the experimental animal is calculated with the following formula:CtmAb,tissue=Ct⁢m⁢A⁢b,tissue,det.Ct⁢i⁢s⁢sue,sample-Cr⁢e⁢f⁢m⁢A⁢b,tissue,det.Ct⁢i⁢s⁢sue,sampleCr⁢e⁢f⁢m⁢A⁢b,plasma.det.*Ct⁢m⁢A⁢b,plasma,det.wherein CtmAb,tissue,det.=obtained by determining the concentration of the therapeutic antibody in the tissue sample of the experimental animal, CtmAb,plasma,det.=obtained by determining the concentration of the therapeutic antibody in a blood sample of the experimental animal directly prior to taking the tissue sample, CrefmAb,tissue,det.=obtained by determining the concentration of the inert reference antibody in the tissue sample of the experimental animal, CrefmAb,plasma,det.=obtained by determining the concentration of an inert reference antibody in the blood sample of the experimental animal directly prior to taking the tissue sample, Ctissue,sampie=obtained by determining the tissue concentration in the tissue sample, whereby the inert reference antibody does not penetrate into said tissue, whereby the inert reference antibody is administered 2 to 10 minutes prior to obtaining the tissue and blood sample.

The current invention is in the field of immunoassays. Morespecifically, herein is reported a method for the determination of theamount of a therapeutic antibody in brain tissue, more specifically ofthe amount of a therapeutic antibody transported across theblood-brain-barrier from the blood into the brain.

BACKGROUND

For the analysis of therapeutic monoclonal antibodies (tmAbs) in samplesof in-vitro or in-vivo origin a respective assay is necessary.

Determining the amount of a therapeutic antibody is generally carriedout by determining the amount of said therapeutic antibody in a sample.Therefore, e.g., an immunoassay, such as, ELISA, RIA, protein blot(Western blot) assay, and the like can be used.

The role of antibodies and their receptors in protection against orderedprotein assembly in neurodegeneration was reviewed by Katsinelos et al.(Front. Immunol. 10 (2019) A1139). They have outlined that IgG levelsare maintained in human serum at around 10 mg/ml. The brain is isolatedfrom serum by the blood-brain barrier (BBB), which is impermeable tolarge macromolecules including IgG and is bathed in cerebrospinal fluid(CSF), which is produced following the filtration of blood and transportof ions across the choroid plexus. Thus, the resulting concentration ofIgG in CSF is around 500- to 1,000-fold lower than in serum.

Hanzatian et al. (mAbs 10 (2018) 765-777) reported that therapeuticmonoclonal antibodies and endogenous IgG antibodies show limited uptakeinto the central nervous system (CNS) due to the blood-brain barrier(BBB), which regulates and controls the selective and specific transportof both exogenous and endogenous materials to the brain. The use ofnatural transport mechanisms, such as receptor-mediated transcytosis(RMT), to deliver antibody therapeutics into the brain have been studiedin rodents and monkeys. After systemic administration of each DVD-Ig,Hanzatian et al. used two independent methods in parallel to observespecific uptake into the brain: An electrochemiluminescent-basedsensitive quantitative assay and a semi-quantitativeimmunohistochemistry technique were used for brain concentrationdetermination and bio distribution/localization in brain, respectively.Significantly enhanced brain uptake and retention was observed for allTfR1 DVD-Ig proteins regardless of the CNS target or the systemicadministration route selected. To prepare brain samples for analysis theused C57BL/6N mice were transcardially perfused with cold Dulbecco'sphosphate-buffered saline (PBS) containing heparin at a rate of 2 ml/minfor 10 min via programmable peristaltic pump.

A comparable approach had been used by Zuchero et al. (Neuron 89 (2016)70-82; wild-type mice, which were IV injected with the target antibodyfollowed by collection of the whole blood and PBS perfusion) andJanowicz et al. (Nature Sci. Rep. 9 (2019) 9255; P301L tau transgenicpR5 mice, to which Alexa-647-labeled IgG, Fab or scFv had beenadministered by retro-orbital injection, were perfused followingtreatment to remove the antibody from their vasculature).

In WO 2018/152359, mice overexpressing human Tau from PS 19 line wereused to evaluate target engagement of chimeric IgG anti-tau antibodyclones 1C7 and 1A1. Therefore, mice were injected i.v. (at 35 mg/kg) ori.p. (at 50 mg/kg) with a control IgG, chimeric IgG clone 1C7, orchimeric IgG clone 1A1. At 2 or 7 days post-injection, cerebral spinalfluid (CSF) was collected via the cistema magna and visually inspectedfor potential blood contamination and following transcardial perfusionwith ice-cold PBS, brain tissue was removed and snap frozen.

Ayabe, M., et al. reported that an anti-human interleukin-6 receptor(hIL-6R) antibody or control antibody were administered intravenously totumor-bearing hIL-6R transgenic mice and bovine serum albumin (BSA) wasadministered intravenously as a marker for residual blood volume intissues. The lysate samples were treated with immune precipitation usinganti-BSA antibody and Protein A magnetic beads followed by trypticdigestion. Each surrogate peptide was analyzed simultaneously byLC/ESI-MS/MS. Corrected tissue concentration was calculated.

Vedeler, et al., reported about immunoglobulins in serum andcerebrospinal fluid from patients with acute Guillain-Barré syndrome(Acta Neurol. Scand. 73 (1986) 388-393.

Shah, et al., reported antibody bio distribution coefficients,especially inferring tissue concentrations of monoclonal antibodiesbased on the plasma concentrations in several preclinical species andhuman (MABS, 5 (2013) 297-305).

Lavezzi, et al., reported MPBPK-TMDD models for mAbs, especiallyalternative models, comparison, and identifiability issues (J.Pharmacokin. Pharmcodyn. 45 (2018) 787-802).

SUMMARY OF THE INVENTION

Herein is reported a method for the determination of the amount of atherapeutic antibody, which has been transported across theblood-brain-barrier from the blood into the brain of an experimentalanimal. The amount is preferably determined in a brain lysate sample.The gist of the invention lies in the additional application of an inertantibody, which is not transported across the blood-brain-barrier,shortly before obtaining the brain sample in which the amount of thetherapeutic antibody transported across the blood-brain-barrier has tobe determined. By applying the inert antibody, a correction value fortherapeutic antibody present in residual blood in the brain sample isobtained. This residual blood-derived amount is used to correct thedetermined amount for non-brain-located antibody. A determinationwithout correction would determine the total amount of therapeuticantibody in the sample, i.e. the amount transported across theblood-brain-barrier into the brain and the amount in residual blood inthe sample. The amount of therapeutic antibody in residual blood is notneglectable, as only about 0.1% of the antibody in the blood will passthe blood-brain-barrier. Thus, the concentration of the therapeuticantibody in the blood exceeds the concentration of the therapeuticantibody in the brain by at least two and up to three orders ofmagnitude. Thereby the results obtained are too high if not correctedwith a method according to the current invention.

The current invention is based, at least in part, on the finding thatfor a robust and correct determination of the amount in brain lysates ofa therapeutic antibody transported across the blood-brain-barrier intothe brain a correction, i.e. reduction, with the amount of therapeuticantibody in residual blood in the brain lysate sample has to be made.

The current invention is based, at least in part, on the finding thatthe amount of residual blood in a brain lysate can be determined byapplying a correction antibody shortly before the brain sample is taken.It has been found that it is especially advantageous to use as referenceantibody an antibody that is not specifically binding to any target inthe experimental animal from which the brain sample is obtained, mostpreferably a human germline antibody.

-   One aspect of the invention is a method/assay for determining the    concentration of a therapeutic antibody in a tissue of an    experimental animal, whereby the tissue has a barrier to the blood    circulation of said animal and whereby the therapeutic antibody had    been administered to said experimental animal, wherein the    interference from residual blood in a tissue sample of the    experimental animal, which is used for determining the concentration    of the therapeutic antibody in said tissue, is reduced, the method    comprising the following steps    -   i) determining the concentration of the therapeutic antibody in        a blood sample of the experimental animal,    -   ii) determining the concentration of the therapeutic antibody in        the tissue sample of the experimental animal,    -   iii) determining the concentration of an inert reference        antibody in the blood sample of the experimental animal,    -   iv) determining the concentration of the inert reference        antibody in the tissue sample of the experimental animal,    -   v) determining the tissue concentration in the tissue sample,    -   and determining the concentration of the therapeutic antibody in        the tissue of the experimental animal with the following        formula:

$\left. {\left. {\left. {\left. {\left. {{C_{{tmAb},{tissue}} = {\frac{C_{{tmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}} - {\frac{\frac{C_{{refmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}}}{C_{{refmAb},{{plasma}.\det.}}}*{C_{{tmAb},{plasma},\det}.{with}}}}}{C_{{tmAb},{plasma},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}i}}} \right){C_{{tmAb},{tissue},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{ii}}}} \right){C_{{refmAb},{tissue},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{iii}}}} \right){C_{{refmAb},{plasma},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{iv}}}} \right){C_{{tissue},{sample}} = {{tissue}{concentration}{of}v}}} \right)$

-   -   -   whereby the inert reference antibody does not cross said            barrier between the tissue and the blood circulation,        -   whereby the inert reference antibody had been            administered i) either together with the therapeutic            antibody in case the sample is to be taken within 5 minutes            after the administration of the therapeutic antibody, or ii)            2 to 10 minutes prior to taking the tissue sample.

-   The same aspect in an alternative wording is, a method for    determining the concentration of a therapeutic antibody in a tissue    of an experimental animal to whom the therapeutic antibody had been    administered, wherein the interference from residual blood in a    tissue sample of the experimental animal, which is used for    determining the concentration of the therapeutic antibody in said    tissue, is reduced,    -   wherein the concentration of the therapeutic antibody in the        tissue of the experimental animal is calculated with the        following formula:

$C_{{tmAb},{tissue}} = {\frac{C_{{tmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}} - {\frac{\frac{C_{{refmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}}}{C_{{refmAb},{{plasma}.\det.}}}*{C_{{tmAb},{plasma},\det}.}}}$

-   -   wherein        -   C_(tmAb,tissue,det.)=obtained by determining the            concentration of the therapeutic antibody in the tissue            sample of the experimental animal,        -   C_(tmAb,plasma,det)=obtained by determining the            concentration of the therapeutic antibody in a blood sample            of the experimental animal,        -   C_(ref,Ab,tissue,det)=obtained by determining the            concentration of the inert reference antibody in the tissue            sample of the experimental animal,        -   C_(refm,Ab,plasma,det.)=obtained by determining the            concentration of an inert reference antibody in the blood            sample of the experimental animal,        -   C_(tisssue,sample)=obtained by determining the tissue            concentration in the tissue sample,        -   whereby the inert reference antibody does not penetrate into            said tissue,        -   whereby the inert reference antibody is administered 2 to 10            minutes prior to obtaining the tissue sample.

The following are all individual embodiment of each and any aspects ofthe invention. Thus, all and any possible permutation of embodiments isdisclosed with respect to any individual aspect according to theinvention,

-   In one embodiment, the blood sample is taken at most 5 minutes prior    to the tissue sample. In one embodiment, the blood sample is taken    prior to the tissue sample. In one embodiment, the blood sample is    taken together or at the same time as the tissue sample.-   In one embodiment, the tissue is either brain tissue and the    therapeutic antibody can cross the blood-brain-barrier or ocular    tissue and the therapeutic antibody can cross the    blood-ocular-barrier.-   One aspect of the invention is a method/assay for determining the    concentration of a therapeutic antibody in brain tissue or a brain    tissue sample of an experimental animal, whereby the brain tissue    has a barrier to the blood circulation of said animal and whereby    the therapeutic antibody had been administered to said experimental    animal, wherein the interference from residual blood in a brain    tissue sample of the experimental animal, which is used for    determining the concentration of the therapeutic antibody in said    brain tissue, is reduced, the method comprising the following steps    -   i) determining the concentration of the therapeutic antibody in        a blood sample of the experimental animal,    -   ii) determining the concentration of the therapeutic antibody in        the brain tissue sample of the experimental animal,    -   iii) determining the concentration of an inert reference        antibody in the blood sample of the experimental animal,    -   iv) determining the concentration of the inert reference        antibody in the brain tissue sample of the experimental animal,    -   v) determining the brain tissue concentration in the tissue        sample,    -   and determining the concentration of the therapeutic antibody in        the brain tissue or the brain tissue sample of the experimental        animal with the following formula:

$\left. {\left. {\left. {\left. {\left. {{C_{{tmAb},{tissue}} = {\frac{C_{{tmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}} - {\frac{\frac{C_{{refmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}}}{C_{{refmAb},{{plasma}.\det.}}}*{C_{{tmAb},{plasma},\det}.{with}}}}}{C_{{tmAb},{plasma},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}i}}} \right){C_{{tmAb},{tissue},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{ii}}}} \right){C_{{refmAb},{tissue},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{iii}}}} \right){C_{{refmAb},{plasma},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{iv}}}} \right){C_{{tissue},{sample}} = {{tissue}{concentration}{of}v}}} \right)$

-   -   -   whereby the inert reference antibody does not cross said            blood-brain-barrier between the brain tissue and the blood            circulation,        -   whereby the inert reference antibody had been            administered i) either together with the therapeutic            antibody in case the brain tissue sample is to be taken            within 5 minutes after the administration of the therapeutic            antibody, or ii) 2 to 10 minutes prior to taking the brain            tissue sample.

-   The same aspect in an alternative wording is, a method for    determining the concentration of a therapeutic antibody in brain    tissue or a brain tissue sample of an experimental animal to whom    the therapeutic antibody had been administered, wherein the    interference from residual blood in the brain tissue sample of the    experimental animal, which is used for determining the concentration    of the therapeutic antibody in said brain tissue, is reduced,    -   wherein the concentration of the therapeutic antibody in the        brain tissue of the experimental animal is calculated with the        following formula:

$C_{{tmAb},{tissue}} = {\frac{C_{{tmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}} - {\frac{\frac{C_{{refmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}}}{C_{{refmAb},{{plasma}.\det.}}}*{C_{{tmAb},{plasma},\det}.}}}$

-   -   wherein        -   C_(tmAb,tissue,det.)=obtained by determining the            concentration of the therapeutic antibody in the brain            tissue sample of the experimental animal,        -   C_(tmAb,plasma,det.)=obtained by determining the            concentration of the therapeutic antibody in a blood sample            of the experimental animal,        -   C_(refmAb,tissue,det)=obtained by determining the            concentration of the inert reference antibody in the brain            tissue sample of the experimental animal,        -   C_(refmAb,plasma,det.)=obtained by determining the            concentration of an inert reference antibody in the blood            sample of the experimental animal,        -   C_(tissue,sample)=obtained by determining the brain tissue            concentration in the brain tissue sample,        -   whereby the inert reference antibody does not penetrate into            said brain tissue,    -   whereby the inert reference antibody is administered 2 to 10        minutes prior to obtaining the brain tissue sample.

-   In one embodiment, the therapeutic antibody is a bispecific    antibody.

-   In one embodiment, the therapeutic antibody is specifically binding    to human transferrin receptor and a brain target.

-   In one embodiment, the brain target is human CD20 or human Abeta or    human alpha-synuclein or human tau or human glucocerebrosidase or    human lingo-1 or human huntingtin.

-   In one embodiment, the experimental animal is selected from mouse,    rat, rabbit, dog, sheep, ape, and monkey.

-   In one embodiment, the experimental animal is a non-human    experimental animal with a body weight of more than 100 g and less    than 15 kg.

-   In one embodiment, the experimental animal is a cynomolgus monkey.

-   In one embodiment, the inert reference antibody is a human germline    antibody.

-   In one embodiment, the inert reference antibody is DP47GS. In one    embodiment, the inert reference antibody comprises a heavy chain    variable domain of SEQ ID NO: 67 and a light chain variable domain    of SEQ ID NO: 68. In one embodiment, the inert reference antibody    comprises a heavy chain of SEQ ID NO: 69 and a light chain of SEQ ID    NO: 70.

-   In one embodiment, the inert reference antibody does not cross said    barrier in detectable amounts within 15 minutes after its    application.

-   In one embodiment, the inert reference antibody does not cross said    barrier in detectable amounts within 10 minutes after its    application.

-   In one embodiment, the inert antibody is administered 5 to 10    minutes prior to taking the tissue sample.

-   In one embodiment, the tissue is perfused with an aqueous solution    directly after taking the blood sample and prior to taking the    tissue sample.

-   In one embodiment, the determining of the concentrations is by a    bridging ELISA.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Herein is reported a method for the determination of the amount of atherapeutic antibody, which has been transported across theblood-brain-barrier from the blood into the brain of an experimentalanimal. The amount is preferably determined in a brain lysate sample.The gist of the invention lies in the additional application of an inertantibody, which is not transported across the blood-brain-barrier,shortly before obtaining the brain sample in which the amount of thetherapeutic antibody transported across the blood-brain-barrier has tobe determined. By applying the inert antibody, a correction value fortherapeutic antibody present in residual blood in the brain sample isobtained. This residual blood-derived amount is used to correct thedetermined amount for non-brain-located antibody. A determinationwithout correction would determine the total amount of therapeuticantibody in the sample, i.e. the amount transported across theblood-brain-barrier into the brain and the amount in residual blood inthe sample. The amount of therapeutic antibody in residual blood is notneglectable, as only about 0.1% of the antibody in the blood will passthe blood-brain-barrier. Thus, the concentration of the therapeuticantibody in the blood exceeds the concentration of the therapeuticantibody in the brain by at least two and up to three orders ofmagnitude. Thereby the results obtained are too high if not correctedwith a method according to the current invention.

The current invention is based, at least in part, on the finding thatfor a robust and correct determination of the amount in brain lysates ofa therapeutic antibody transported across the blood-brain-barrier intothe brain a correction, i.e. reduction, with the amount of therapeuticantibody in residual blood in the brain lysate sample has to be made.

The current invention is based, at least in part, on the finding thatthe amount of residual blood in a brain lysate can be determined byapplying a correction antibody shortly before the brain sample is taken.It has been found that it is especially advantageous to use as referenceantibody an antibody that is not specifically binding to any target inthe experimental animal from which the brain sample is obtained, mostpreferably a human germline antibody.

-   One aspect of the invention is a method/assay for determining the    concentration of a therapeutic antibody in a tissue of an    experimental animal, whereby the tissue has a barrier to the blood    circulation of said animal and whereby the therapeutic antibody had    been administered to said experimental animal, wherein the    interference from residual blood in a tissue sample of the    experimental animal, which is used for determining the concentration    of the therapeutic antibody in said tissue, is reduced, the method    comprising the following steps    -   i) determining the concentration of the therapeutic antibody in        a blood serum sample of the experimental animal,    -   ii) determining the concentration of the therapeutic antibody in        the tissue sample of the experimental animal,    -   iii) determining the concentration of an inert reference        antibody in the blood serum sample of the experimental animal,    -   iv) determining the concentration of the inert reference        antibody in the tissue sample of the experimental animal,    -   v) determining the tissue concentration in the tissue sample,    -   and determining the concentration of the therapeutic antibody in        the tissue of the experimental animal with the following        formula:

$\left. {\left. {\left. {\left. {\left. {{C_{{tmAb},{tissue}} = {\frac{C_{{tmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}} - {\frac{\frac{C_{{refmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}}}{C_{{refmAb},{{plasma}.\det.}}}*{C_{{tmAb},{plasma},\det}.{with}}}}}{C_{{tmAb},{plasma},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}i}}} \right){C_{{tmAb},{tissue},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{ii}}}} \right){C_{{refmAb},{tissue},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{iii}}}} \right){C_{{refmAb},{plasma},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{iv}}}} \right){C_{{tissue},{sample}} = {{tissue}{concentration}{of}v}}} \right)$

-   -   -   whereby the inert reference antibody does not cross said            barrier between the tissue and the blood circulation,        -   whereby the inert reference antibody had been            administered i) either together with the therapeutic            antibody in case the sample is to be taken within 5 minutes            after the administration of the therapeutic antibody, or ii)            2 to 10 minutes prior to taking the tissue sample,        -   whereby the blood sample is taken together/directly prior/at            the same time as the tissue sample.

Definitions

The knobs into holes dimerization modules and their use in antibodyengineering are described in Carter P.; Ridgway J. B. B.; Presta L. G.:Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73(1).

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and is referred to as“numbering according to Kabat” herein. Specifically, the Kabat numberingsystem (see pages 647-660) of Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) is used for the light chainconstant domain CL of kappa and lambda isotype, and the Kabat EU indexnumbering system (see pages 661-723) is used for the constant heavychain domains (CH1, Hinge, CH2 and CH3, which is herein furtherclarified by referring to “numbering according to Kabat EU index” inthis case).

The term “about” denotes a range of +/−20% of the thereafter followingnumerical value. In one embodiment, the term about denotes a range of+/−10% of the thereafter-following numerical value. In one embodiment,the term about denotes a range of +/−5% of the thereafter-followingnumerical value.

The term “antibody-dependent cellular cytotoxicity (ADCC)” is a functionmediated by Fc receptor binding and refers to lysis of target cells byan antibody as reported herein in the presence of effector cells. ADCCis measured in one embodiment by the treatment of a preparation of CD19expressing erythroid cells (e.g. K562 cells expressing recombinant humanCD19) with an antibody as reported herein in the presence of effectorcells such as freshly isolated PBMC (peripheral blood mononuclear cells)or purified effector cells from buffy coats, like monocytes or NK(natural killer) cells. Target cells are labeled with 51Cr andsubsequently incubated with the antibody. The labeled cells areincubated with effector cells and the supernatant is analyzed forreleased 51Cr. Controls include the incubation of the target endothelialcells with effector cells but without the antibody. The capacity of theantibody to induce the initial steps mediating ADCC is investigated bymeasuring their binding to Fcγ receptors expressing cells, such ascells, recombinantly expressing FcγRI and/or FcγRIIA or NK cells(expressing essentially FcγRIIIA) In one preferred embodiment, bindingto FcγR on NK cells is measured.

The term “amplifier” denotes an entity or process that enhances thesignal in a detection method, such as an ELISA (e.g., an enzymaticamplifier used in an ELISA).

The terms “anti-human A-beta antibody” and “an antibody specificallybinding to human A-beta” refer to an antibody that is capable of bindingthe human A-beta peptide with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting A-betapeptide.

It is of note that human A-beta has several naturally occurring forms,whereby the human forms are referred to as Aβ39, Aβ40, Aβ41, Aβ42 andAβ43. The most prominent form, Aβ42, has the amino acid sequence of SEQID NO: 01. In Aβ41, Aβ40, Aβ39, the C-terminal amino acids A, IA and VIAare missing, respectively. In the Aβ43 form, an additional threonineresidue is comprised at the C-terminus of SEQ ID NO: 01 (33106).

Thus, the term also encompasses antibodies that bind to a shortenedfragment of the human A-beta polypeptide.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, or multispecific antibodies (e.g.,bispecific antibodies).

An antibody in general comprises two so called light chain polypeptides(light chain) and two so called heavy chain polypeptides (heavy chain).Each of the heavy and light chain polypeptides contains a variabledomain (variable region) (generally the amino terminal portion of thepolypeptide chain) comprising binding regions that are able to interactwith an antigen. Each of the heavy and light chain polypeptidescomprises a constant region (generally the carboxyl terminal portion).The constant region of the heavy chain mediates the binding of theantibody i) to cells bearing a Fc gamma receptor (FcγR), such asphagocytic cells, or ii) to cells bearing the neonatal Fc receptor(FcRn) also known as Brambell receptor. It also mediates the binding tosome factors including factors of the classical complement system suchas component (C1q). The constant domains of an antibody heavy chaincomprise the CH1-domain, the CH2-domain and the CH3-domain, whereas thelight chain comprises only one constant domain, CL, which can be of thekappa isotype or the lambda isotype.

The variable domain of an immunoglobulin's light or heavy chain in turncomprises different segments, i.e. four framework regions (FR) and threehypervariable regions (HVR).

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv, and scFab); single domain antibodies (dAbs); andmultispecific antibodies formed from antibody fragments. For a review ofcertain antibody fragments, see Holliger and Hudson, NatureBiotechnology 23:1126-1136 (2005).

The “blood-brain-barrier” or “BBB” refers to the physiological barrierbetween the peripheral circulation and the brain and spinal cord, whichis formed by tight junctions within the brain capillary endothelialplasma membranes, creating a tight barrier that, restricts the transportof molecules into the brain, even very small molecules such as urea (60Daltons). The BBB within the brain, the blood-spinal-cord barrier withinthe spinal cord, and the blood-retinal-barrier within the retina arecontiguous capillary barriers within the CNS, and are hereincollectively referred to an the blood-brain-barrier or BBB. The BBB alsoencompasses the blood-CSF barrier (choroid plexus) where the barrier iscomprised of ependymal cells rather than capillary endothelial cells.

A “blood-brain-barrier receptor” (abbreviated “BBBR” herein) is anextracellular membrane-linked receptor protein expressed on brainendothelial cells which is capable of transporting molecules across theBBB or be used to transport exogenous administrated molecules. Examplesof BBBR herein include: transferrin receptor (TfR), insulin receptor,insulin-like growth factor receptor (IGF-R), low density lipoproteinreceptors including without limitation low density lipoproteinreceptor-related protein 1 (LRP1) and low density lipoproteinreceptor-related protein 8 (LRP8), and heparin-binding epidermal growthfactor-like growth factor (HB-EGF). One preferred BBBR is transferrinreceptor (TfR).

The term “brain effector entity” denotes a molecule that is to betransported to the brain across the BBB. The effector entity typicallyhas a characteristic therapeutic activity that is desired to bedelivered to the brain. Effector entities include neurologicallydisorder drugs and cytotoxic agents such as e.g. polypeptides andantibodies, in particular monoclonal antibodies or fragments thereofdirected to a brain target.

The term “capture antibody” denotes an antibody that is used in asandwich ELISA format to bind (i.e., capture) a target substance presentin a sample for detection. A second antibody (i.e., the detectionantibody) then binds to the captured target and allows detection of theantibody-target-antibody-complex (forming a “sandwich” comprised ofantibody-target-antibody).

The “central nervous system” or “CNS” refers to the complex of nervetissues that control bodily function, and includes the brain and spinalcord.

The terms “CNS antigen” and “brain target” denote an antigen and/ormolecule expressed in the CNS, including the brain, which can betargeted with an antibody or small molecule. Examples of such antigenand/or molecule include, without limitation: beta-secretase 1 (BACE1),amyloid beta (Abeta), epidermal growth factor receptor (EGFR), humanepidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4(ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucinerich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gammasecretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75neurotrophin receptor (p75NTR), glucocerebrosidase and caspase 6.

A “conjugate” is fusion protein of the present invention conjugated toone or more heterologous molecule(s), including but not limited to alabel, neurological disorder drug or cytotoxic agent.

The term “detection antibody” denotes an antibody, which carries a meansfor visualization or quantitation. Such a means is typically an enzyme(catalyzing a colored or fluorescent reaction product following theaddition of a suitable substrate), such as, e.g., horseradishperoxidase, urease, alkaline phosphatase, glucoamylase andβ-galactosidase. In some embodiments, the detection antibody is directedagainst the antigen of interest. In some embodiments, the detectionantibody is an anti-species antibody. In some embodiments, the detectionantibody is conjugated to a detectable label such as biotin, afluorescent marker, or a radioisotope, and is detected and/orquantitated using this label.

The term “detection reagent” denotes a reagent, which permits thedetection and/or quantitation of an antibody, bound to an antigen. Insome embodiments, the detection reagent is a colorimetric substrate foran enzyme that has been conjugated to an antibody. Addition of asuitable substrate to the antibody-enzyme conjugate results in theproduction of a colorimetric or fluorimetric signal (e.g., following thebinding of the conjugated antibody to the antigen of interest). Thisdefinition also encompasses the use of biotin and avidin-based compounds(e.g., including but not limited to neutravidin and streptavidin) aspart of the detection system.

The term “directly after” as used herein denotes the time span betweentaking a first sample and a second sample, which only encompasses thechange of the sampling device and the actual time for taking the sample.In one embodiment, the term directly after denotes a time period of 5minutes or less, in a further embodiment, of 3 minutes or less, in onepreferred embodiment, of 2 minutes or less.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody class.Examples of antibody effector functions include C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

Fc receptor binding dependent effector functions can be mediated by theinteraction of the Fc-region of an antibody with Fc receptors (FcRs),which are specialized cell surface receptors on hematopoietic cells. Fcreceptors belong to the immunoglobulin superfamily, and have been shownto mediate both the removal of antibody-coated pathogens by phagocytosisof immune complexes, and the lysis of erythrocytes and various othercellular targets (e.g. tumor cells) coated with the correspondingantibody, via antibody dependent cell mediated cytotoxicity (ADCC) (seee.g. Van de Winkel, J. G. and Anderson, C. L., J. Leukoc. Biol. 49(1991) 511-524). FcRs are defined by their specificity forimmunoglobulin isotypes: Fc receptors for IgG antibodies are referred toas FcγR. Fc receptor binding is described e.g. in Ravetch, J. V. andKinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., etal., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin.Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76(1998) 231-248.

Cross-linking of receptors for the Fc-region of IgG antibodies (FcγR)triggers a wide variety of effector functions including phagocytosis,antibody-dependent cellular cytotoxicity, and release of inflammatorymediators, as well as immune complex clearance and regulation ofantibody production. In humans, three classes of FcγR have beencharacterized, which are:

-   -   FcγRI (CD64) binds monomeric IgG with high affinity and is        expressed on macrophages, monocytes, neutrophils and        eosinophils. Modification in the Fc-region IgG at least at one        of the amino acid residues E233-G236, P238, D265, N297, A327 and        P329 (numbering according to EU index of Kabat) reduce binding        to FcγRI. IgG2 residues at positions 233-236, substituted into        IgG1 and IgG4, reduced binding to FcγRI by 10³-fold and        eliminated the human monocyte response to antibody-sensitized        red blood cells (Armour, K. L., et al., Eur. J. Immunol.        29 (1999) 2613-2624).    -   FcγRII (CD32) binds complexed IgG with medium to low affinity        and is widely expressed. This receptor can be divided into two        sub-types, FcγRIIA and FcγRIIB FcγRIIA is found on many cells        involved in killing (e.g. macrophages, monocytes, neutrophils)        and seems able to activate the killing process. FcγRIIB seems to        play a role in inhibitory processes and is found on B-cells,        macrophages and on mast cells and eosinophils. On B-cells, it        seems to function to suppress further immunoglobulin production        and isotype switching to, for example, the IgE class. On        macrophages, FcγRIIB acts to inhibit phagocytosis as mediated        through FcγRIIA. On eosinophils and mast cells, the B-form may        help to suppress activation of these cells through IgE binding        to its separate receptor. Reduced binding for FcγRIIA is found        e.g. for antibodies comprising an IgG Fc-region with mutations        at least at one of the amino acid residues E233-G236, P238,        D265, N297, A327, P329, D270, Q295, A327, R292, and K414        (numbering according to EU index of Kabat).    -   FcγRIII (CD16) binds IgG with medium to low affinity and exists        as two types. FcγRIIIA is found on NK cells, macrophages,        eosinophils and some monocytes and T cells and mediates ADCC.        FcγRIIIB is highly expressed on neutrophils. Reduced binding to        FcγRIIIA is found e.g. for antibodies comprising an IgG        Fc-region with mutation at least at one of the amino acid        residues E233-G236, P238, D265, N297, A327, P329, D270, Q295,        A327, 5239, E269, E293, Y296, V303, A327, K338 and D376        (numbering according to EU index of Kabat).

Mapping of the binding sites on human IgG1 for Fc receptors, the abovementioned mutation sites and methods for measuring binding to FcγRT andFcγRIIA are described in Shields, R. L., et al. J. Biol. Chem. 276(2001) 6591-6604.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “ELISA” denotes an enzyme-linked immunosorbent assay. DifferentELISA formats and applications are known in the art (see, e.g.,Crowther, “Enzyme-Linked Immunosorbent Assay (ELISA),” in MolecularBiomethods Handbook, Rapley et al. [eds.], pp. 595-617, Humana Press,Inc., Totowa, N.J. (1998); Harlow and Lane (eds.), Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1988); Ausubelet al. (eds.), Current Protocols in Molecular Biology, Ch. 11, JohnWiley & Sons, Inc., New York (1994)).

One specific ELISA format is a so-called “direct ELISA”. In this ELISAformat a target, e.g. a polypeptide, present in a sample is detected. Ina direct ELISA the sample, containing the target, is brought in contactwith a solid phase, such as e.g., stationary or immobilized support(e.g., a microtiter plate well). The target, if present in the sample,becomes immobilized to the solid phase, and is thereafter detecteddirectly using an enzyme-conjugated detection molecule. If the target isan antigen the detection molecule is an antibody specific for theantigen, or if the target is an antibody specific for an antigen thedetection molecule is an enzyme-conjugated antibody specific for theantigen.

Another specific ELISA format is a so-called “indirect ELISA”. In thisELISA format, an antigen (or an antibody) is immobilized to a solidphase (e.g., a microtiter plate well). Thereafter an antigen-specificantibody (or antigen) is added followed by the addition of a detectionantibody specific for the antibody that specifically binds the antigen.This detection antibody can be a “species-specific” antibody (e.g., agoat anti-rabbit antibody).

Another specific ELISA format is a so-called “sandwich ELISA”. In thisformat the antigen is immobilized on a solid phase (e.g., a microtiterplate well) via capture by an antibody specifically binding to theantigen (i.e., a capture antibody), which is (covalently or via aspecific binding pair) immobilized on the solid phase. Generally, asample comprising the antigen is added to the solid phase, followed bywashing. If the antigen of interest is present in the sample, it isbound by the capture antibody to the solid phase.

The above-specified ELISA formats can be combined. A sandwich ELISA canbe a “direct sandwich ELISA”, wherein the captured antigen is detecteddirectly by using an enzyme-conjugated antibody directed against theantigen. A sandwich ELISA can be an “indirect sandwich ELISA”, whereinthe captured antigen is detected indirectly by using an antibodydirected against the antigen, which is then detected by anotherenzyme-conjugated antibody which binds the antigen-specific antibodyeither directly or via an attached label. With a reporter reagent, thethird antibody is detected.

The term “Fc receptor” as used herein refers to activation receptorscharacterized by the presence of a cytoplasmic ITAM sequence associatedwith the receptor (see e.g. Ravetch, J. V. and Bolland, S., Annu. Rev.Immunol. 19 (2001) 275-290). Such receptors are FcγRI, FcγRIIA andFcγRIIIA The term “no binding of FcγR” denotes that at an antibodyconcentration of 10 μg/ml the binding of an antibody as reported hereinto NK cells is 10% or less of the binding found for anti-OX40L antibodyLC.001 as reported in WO 2006/029879.

While IgG4 shows reduced FcR binding, antibodies of other IgG subclassesshow strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fccarbohydrate), Pro329 and 234, 235, 236 and 237 Ile253, Ser254, Lys288,Thr307, Gln311, Asn434, and His435 are residues which provide if alteredalso reduce FcR binding (Shields, R. L., et al. J. Biol. Chem. 276(2001) 6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan,A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434). In oneembodiment, the antibody as reported herein is of IgG1 or IgG2 subclassand comprises the mutation PVA236, GLPSS331, and/or L234A/L235A. In oneembodiment, the antibody as reported herein is of IgG4 subclass andcomprises the mutation L235E. In one embodiment, the antibody furthercomprises the mutation S228P.

The term “Fc-region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication 91-3242.

The antibodies as reported herein comprise as Fc-region, in oneembodiment, an Fc-region derived from human origin. In one embodiment,the Fc-region comprises all parts of the human constant region. TheFc-region of an antibody is directly involved in complement activation,C1q binding, C3 activation and Fc receptor binding. While the influenceof an antibody on the complement system is dependent on certainconditions, binding to C1q is caused by defined binding sites in theFc-region. Such binding sites are known in the state of the art anddescribed e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16 (1979)907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E. E., et al.,J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75(2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324;and EP 0 307 434. Such binding sites are e.g. L234, L235, D270, N297,E318, K320, K322, P331 and P329 (numbering according to EU index ofKabat; Unless otherwise specified herein, numbering of amino acidresidues in the Fc-region or constant region is according to the EUnumbering system, also called the EU index, as described in Kabat, E. A.et al., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991), NIHPublication 91-3242). Antibodies of subclass IgG1, IgG2 and IgG3 usuallyshow complement activation, C1q binding and C3 activation, whereas IgG4do not activate the complement system, do not bind C1q and do notactivate C3. An “Fc-region of an antibody” is a term well known to theskilled artisan and defined on the basis of papain cleavage ofantibodies. In one embodiment, the Fc-region is a human Fc-region. Inone embodiment, the Fc-region is of the human IgG4 subclass comprisingthe mutations S228P and/or L235E (numbering according to EU index ofKabat). In one embodiment, the Fc-region is of the human IgG1 subclasscomprising the mutations L234A and L235A (numbering according to EUindex of Kabat).

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure, i.e.comprising two light chains and two heavy chains.

A “human antibody” is one, which possesses an amino acid sequence, whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “in-vitro” denotes either an artificial environment as such orthat a process or reaction is performed within such an artificialenvironment.

The term “in-vivo” denotes the natural environment (e.g., an animal or acell) of a compound or that a process or reaction is performed withinits natural environment.

The term “immunoassay” denotes any technique that utilizes specificallybinding molecules, such as antibodies, to capture and/or detect aspecific target for qualitative or quantitative analysis. In general, animmunoassay is characterized by the following steps: 1) immobilizationor capture of the analyte and 2) detection and measuring the analyte.The analyte can be captured, i.e. bound, on any solid surface, such ase.g. a membrane, plastic plate, or some other solid surface.

The term “linker” denotes a chemical linker or a single chain peptidiclinker that covalently connects different entities of theblood-brain-barrier shuttle module and/or the fusion polypeptide and/orthe conjugate as reported herein. The linker connects for example thebrain effector entity to the monovalent binding entity. For example, ifthe monovalent binding entity comprises a CH2-CH3 Ig entity and a scFabdirected to the blood-brain-barrier-receptor, then the linker conjugatesthe scFab to the C-terminal end of the CH3-CH2 Ig entity. The linkerconjugating the brain effector entity to the monovalent binding entity(first linker) and the linker connecting the scFab to the C-terminal endof the CH2-CH3 Ig domain (second linker) can be the same or different.

Single chain peptidic linkers, comprising of from one to twenty aminoacid residues joined by peptide bonds, can be used. In certainembodiments, the amino acids are selected from the twenty naturallyoccurring amino acids. In certain other embodiments, one or more of theamino acids are selected from glycine, alanine, proline, asparagine,glutamine and lysine. In other embodiments, the linker is a chemicallinker. In certain embodiments, the linker is a single chain peptidiclinker with an amino acid sequence with a length of at least 25 aminoacid residues, in one preferred embodiment, with a length of 32 to 50amino acid residues. In one embodiment, the peptidic linker is a (G×S)nlinker with G=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6, 7or 8), in one embodiment, with x=4, n=6 or 7, in one preferredembodiment, with x=4, n=7. In one embodiment, the linker is (G4S)4 (SEQID NO: 02). In one embodiment, the linker is (G4S)6G2 (SEQ ID NO: 03).

Conjugation may be performed using a variety of chemical linkers. Forexample, the monovalent binding entity or the fusion polypeptide and thebrain effector entity may be conjugated using a variety of bifunctionalprotein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctionalderivatives of imidoesters (such as dimethyl adipimidate HC1), activeesters (such as disuccinimidyl suberate), aldehydes (such asglutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). The linker may be a “cleavable linker”facilitating release of the effector entity upon delivery to the brain.For example, an acid-labile linker, peptidase-sensitive linker,photo-labile linker, dimethyl linker or disulfide-containing linker(Chari et al, Cancer Res. 52 (1992) 127-131; U.S. Pat. No. 5,208,020)may be used.

Covalent conjugation can either be direct or via a linker. In certainembodiments, direct conjugation is by construction of a polypeptidefusion (i.e. by genetic fusion of the two genes encoding the monovalentbinding entity towards the BBBR and effector entity and expressed as asingle polypeptide (chain)). In certain embodiments, direct conjugationis by formation of a covalent bond between a reactive group on one ofthe two portions of the monovalent binding entity against the BBBR and acorresponding group or acceptor on the brain effector entity. In certainembodiments, direct conjugation is by modification (i.e. geneticmodification) of one of the two molecules to be conjugated to include areactive group (as non-limiting examples, a sulfhydryl group or acarboxyl group) that forms a covalent attachment to the other moleculeto be conjugated under appropriate conditions. As one non-limitingexample, a molecule (i.e. an amino acid) with a desired reactive group(i.e. a cysteine residue) may be introduced into, e.g., the monovalentbinding entity towards the BBBR antibody and a disulfide bond formedwith the neurological therapeutic antibody. Methods for covalentconjugation of nucleic acids to proteins are also known in the art(i.e., photo-crosslinking, see, e.g., Zatsepin et al. Russ. Chem. Rev.74 (2005) 77-95). Conjugation may also be performed using a variety oflinkers. For example, a monovalent binding entity and a effector entitymay be conjugated using a variety of bifunctional protein couplingagents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HC1), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Peptidic linkers, comprised of fromone to twenty amino acid residues joined by peptide bonds, may also beused. In certain such embodiments, the amino acid residues are selectedfrom the twenty naturally occurring amino acids. In certain other suchembodiments, one or more of the amino acid residues are selected fromglycine, alanine, proline, asparagine, glutamine and lysine. The linkermay be a “cleavable linker” facilitating release of the effector entityupon delivery to the brain. For example, an acid-labile linker,peptidase-sensitive linker, photo-labile linker, dimethyl linker ordisulfide-containing linker (Chari et al, Cancer Res. 52 (1992) 127-131;U.S. Pat. No. 5,208,020) may be used.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identical,except for possible variant antibodies, e.g., containing naturallyoccurring mutations or arising during production of a monoclonalantibody preparation, such variants generally being present in minoramounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. Thus, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies in accordance with the present invention may be made by avariety of techniques, including but not limited to the hybridomamethod, recombinant DNA methods, phage-display methods, and methodsutilizing transgenic animals containing all or part of the humanimmunoglobulin loci, such methods and other exemplary methods for makingmonoclonal antibodies being described herein.

The term “monovalent binding entity” denotes a molecule able to bindspecifically and in a monovalent binding mode to a BBBR. The blood brainshuttle module and/or conjugate as reported herein are characterized bythe presence of a single unit of a monovalent binding entity i.e. theblood brain shuttle module and/or conjugate of the present inventioncomprise exactly one unit of the monovalent binding entity. Themonovalent binding entity includes but is not limited to polypeptides,full length antibodies, antibody fragments including Fab, Fab′, Fvfragments, single-chain antibody molecules such as e.g. single chainFab, scFv. The monovalent binding entity can for example be a scaffoldprotein engineered using state of the art technologies like phagedisplay or immunization. The monovalent binding entity can also be apolypeptide. In certain embodiments, the monovalent binding entitycomprises a CH2-CH3 Ig domain and a single chain Fab (scFab) directed toa blood-brain-barrier-receptor. The scFab is coupled to the C-terminalend of the CH2-CH3 Ig domain by a linker. In certain embodiments, thescFab is directed to the transferrin receptor.

The term “monovalent binding mode” denotes a specific binding to theBBBR where the interaction between the monovalent binding entity and theBBBR takes place through one single epitope. The monovalent binding modeprevents any dimerization/multimerization of the BBBR due to a singleepitope interaction point. The monovalent binding mode prevents that theintracellular sorting of the BBBR is altered.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical composition.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 Daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variabledomain (VH), also called a variable heavy domain or a heavy chainvariable region, followed by three constant heavy domains (CH1, CH2, andCH3). Similarly, from N- to C-terminus, each light chain has a variabledomain (VL), also called a variable light domain or a light chainvariable region, followed by a constant light (CL) domain.

The term “pharmaceutical composition” or “pharmaceutical formulation”refers to a preparation which is in such form as to permit thebiological activity of an active ingredient contained therein to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the pharmaceutical compositionwould be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition or formulation, other than an activeingredient, which is nontoxic to a subject. A pharmaceuticallyacceptable carrier includes, but is not limited to, a buffer, excipient,stabilizer, or preservative.

The term “sample” includes, but is not limited to, any quantity of asubstance from a living thing or formerly living thing. Such livingthings include mice, monkeys, rats, rabbits, and other animals. In oneembodiment, the sample is obtained from a monkey, especially acynomolgus monkey, or a rabbit, or a mouse, or a rat.

The term “signal” as used herein encompasses any detectable physicalchange that can be used to indicate that a reaction has taken place, forexample, binding of an antibody to its antigen. It is contemplated thatsignals in the form of fluorimetric or colorimetric products/reagentsare specific forms of a signal and can be used in the method accordingto the current invention. In some embodiments of the present invention,the signal is assessed quantitatively.

The term “solid phase” denotes a non-fluid substance, and includesparticles (including microparticles and beads) made from materials suchas polymer, metal (paramagnetic, ferromagnetic particles), glass, andceramic; gel substances such as silica, alumina, and polymer gels;capillaries, which may be made of polymer, metal, glass, and/or ceramic;zeolites and other porous substances; electrodes; microtiter plates;solid strips; and cuvettes, tubes or other spectrometer samplecontainers. A solid phase component is distinguished from inert solidsurfaces in that a “solid phase” contains at least one moiety on itssurface, which is intended to interact with a substance in a sample. Asolid phase may be a stationary component, such as a tube, strip,cuvette or microtiter plate, or may be non-stationary components, suchas beads and microparticles. A variety of microparticles that alloweither non-covalent or covalent attachment of proteins and othersubstances may be used. Such particles include polymer particles such aspolystyrene and poly (methyl methacrylate); gold particles such as goldnanoparticles and gold colloids; and ceramic particles such as silica,glass, and metal oxide particles. See for example Martin, C. R., et al.,Analytical Chemistry-News & Features, 70 (1998) 322A-327A, or Butler, J.E., Methods 22 (2000) 4-23.

The terms “therapeutic (monoclonal) antibody” and “drug” are usedinterchangeably herein. These terms are used in the broadest sense andencompasses various antibody structures, including but not limited tomonoclonal antibodies, polyclonal antibodies, and antibody fragments solong as they exhibit the desired antigen-binding activity.

The “transferrin receptor” (“TfR”) is a transmembrane glycoprotein (witha molecular weight of about 180,000 Da) composed of twodisulphide-bonded sub-units (each of apparent molecular weight of about90,000 Da) involved in iron uptake in vertebrates. In one embodiment,the TfR as mentioned herein is human TfR comprising the amino acidsequence as in Schneider et al (Nature 311 (1984) 675-678), for example.

Multispecific Antibodies

In certain embodiments, the therapeutic antibody is a bispecificantibody. In one embodiment, the therapeutic antibody is a bispecific,trivalent antibody. In one preferred embodiment, the therapeuticantibody is a monoclonal, bispecific, trivalent antibody.

In certain embodiments, the therapeutic antibody is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent antigens. In certain embodiments, one of the bindingspecificities is for a first antigen and the other is for a differentsecond antigen. Bispecific antibodies can be prepared as full-lengthantibodies or antibody fragments. In one embodiment, the antibody is abispecific antibody, which specifically binds to a first and a secondantigen. In one embodiment, the bispecific antibody has i) a firstbinding specificity that specifically binds to a first antigen, and ii)a second binding specificity that specifically binds to a secondantigen. In one embodiment, the antibody is a bispecific, trivalentantibody. In one preferred embodiment, the antibody is a monoclonal,bispecific, trivalent antibody.

In one embodiment, one of the binding sites specifically binds to aBBBR.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A., et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A., et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M., et al.,J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodiesas described, e.g., in Tutt, A., et al., J. Immunol. 147 (1991) 60-69).

Multispecific antibodies are described in WO 2009/080251, WO2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO2010/115589, WO 2010/136172, WO 2010/145792, or WO 2010/145793.

Different bispecific antibody formats are known.

Exemplary bispecific antibody formats for which the methods as reportedherein can be used are

-   -   the CrossMab format (=CrossMab): a multispecific IgG antibody        comprising a first Fab fragment and a second Fab fragment,        wherein in the first Fab fragment        -   a) only the CH1 and CL domains are replaced by each other            (i.e. the light chain of the first Fab fragment comprises a            VL and a CH1 domain and the heavy chain of the first Fab            fragment comprises a VH and a CL domain);        -   b) only the VH and VL domains are replaced by each other            (i.e. the light chain of the first Fab fragment comprises a            VH and a CL domain and the heavy chain of the first Fab            fragment comprises a VL and a CH1 domain); or        -   c) the CH1 and CL domains are replaced by each other and the            VH and VL domains are replaced by each other (i.e. the light            chain of the first Fab fragment comprises a VH and a CH1            domain and the heavy chain of the first Fab fragment            comprises a VL and a CL domain); and    -   wherein the second Fab fragment comprises a light chain        comprising a VL and a CL domain, and a heavy chain comprising a        VH and a CH1 domain;    -   the CrossMab may comprises a first heavy chain including a CH3        domain and a second heavy chain including a CH3 domain, wherein        both CH3 domains are engineered in a complementary manner by        respective amino acid substitutions, in order to support        heterodimerization of the first heavy chain and the modified        second heavy chain, e.g. as disclosed in WO 96/27011, WO        98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO        2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO        2012/058768, WO 2013/157954, or WO 2013/096291 (incorporated        herein by reference);    -   the one-armed single chain format (=one-armed single chain        antibody): antibody comprising a first binding site that        specifically binds to a first antigen and a second binding site        that specifically binds to a second antigen, whereby the        individual chains are as follows        -   light chain (variable light chain domain+light chain kappa            constant domain)        -   combined light/heavy chain (variable light chain            domain+light chain constant domain+peptidic linker+variable            heavy chain domain+CH1+Hinge+CH2+CH3 with knob mutation)        -   heavy chain (variable heavy chain domain+CH1+Hinge+CH2+CH3            with hole mutation);    -   the two-armed single chain format (=two-armed single chain        antibody): antibody comprising a first binding site that        specifically binds to a first antigen and a second binding site        that specifically binds to a second antigen, whereby the        individual chains are as follows        -   combined light/heavy chain 1 (variable light chain            domain+light chain constant domain+peptidic linker+variable            heavy chain domain+CH1+Hinge+CH2+CH3 with hole mutation)        -   combined light/heavy chain 2 (variable light chain            domain+light chain constant domain+peptidic linker+variable            heavy chain domain+CH1+Hinge+CH2+CH3 with knob mutation);    -   the common light chain bispecific format (=common light chain        bispecific antibody): antibody comprising a first binding site        that specifically binds to a first antigen and a second binding        site that specifically binds to a second antigen, whereby the        individual chains are as follows        -   light chain (variable light chain domain+light chain            constant domain)        -   heavy chain 1 (variable heavy chain domain+CH1+Hinge+CH2+CH3            with hole mutation)        -   heavy chain 2 (variable heavy chain domain+CH1+Hinge+CH2+CH3            with knob mutation);    -   the bispecific Fab format: Fab comprising two (non-overlapping)        paratopes in a complementary pair of a VH and a VL domain,        wherein the first paratope comprises (consists of) amino acid        residues from CDR1 and CDR3 of the VL domain and CDR2 of the VH        domain, and the second paratope comprises (consists of) residues        from CDR1 and CDR3 of the VH domain and CDR2 of the VL domain;        the term “non-overlapping” in this context indicates that an        amino acid residue that is comprised within the first paratope        of the bispecific Fab is not comprised in the second paratope,        and an amino acid that is comprised within the second paratope        of the bispecific Fab is not comprised in the first paratope;    -   the TCB format: a bispecific antibody comprising        -   a first and a second Fab fragment, wherein each binding site            of the first and the second Fab fragment specifically bind            to a second antigen,        -   a third Fab fragment, wherein the binding site of the third            Fab fragment specifically binds to a first antigen, and            wherein the third Fab fragment comprises a domain crossover            such that the variable light chain domain (VL) and the            variable heavy chain domain (VH) are replaced by each other,            and        -   an Fc-region comprising a first Fc-region polypeptide and a            second Fc-region polypeptide,    -   wherein the first and the second Fab fragment each comprise a        heavy chain fragment and a full-length light chain,    -   wherein the C-terminus of the heavy chain fragment of the first        Fab fragment is fused to the N-terminus of the first Fc-region        polypeptide,    -   wherein the C-terminus of the heavy chain fragment of the second        Fab fragment is fused to the N-terminus of the variable light        chain domain of the third Fab fragment and the C-terminus of the        heavy chain constant domain 1 of the third Fab fragment is fused        to the N-terminus of the second Fc-region polypeptide.    -   the brain-shuttle format (BS): a bispecific antibody comprising        -   a) one (full length) antibody comprising two pairs each of a            (full length) antibody light chain and a (full length)            antibody heavy chain, wherein the binding sites formed by            each of the pairs of the (full length) heavy chain and the            (full length) light chain specifically bind to a first            antigen, and        -   b) one additional Fab fragment, wherein the additional Fab            fragment is fused to any C-terminus of one heavy chain of            the (full length) antibody, wherein the binding site of the            additional Fab fragment specifically binds to a second            antigen,        -   wherein the additional Fab fragment specifically binding to            the second antigen comprises a domain crossover such that            the constant light chain domain (CL) and the constant heavy            chain domain 1 (CH1) are replaced by each other, and        -   wherein the first antigen is the brain target and the second            antigen is human transferrin receptor.

In one embodiment, the bispecific antibody is a CrossMab.

In one embodiment, the bispecific antibody is a one-armed single chainantibody.

In one embodiment, the bispecific antibody is a two-armed single chainantibody.

In one embodiment, the bispecific antibody is a common light chainbispecific antibody.

In one embodiment, the bispecific antibody is a bispecific Fab.

In one embodiment, the bispecific antibody is a TCB.

In one embodiment, the bispecific antibody is a BS.

Multivalent, multispecific antibodies specifically bind to differenttargets, most likely with different affinities and complex stabilitiesfor each target. Only a fully active multivalent, multispecific antibodycan bind to all targets and shows the full biological activity in acorresponding assay.

A. Exemplary Bispecific Antibody: Anti-Human A-Beta/Human TransferrinReceptor Antibody

In certain embodiments, the therapeutic antibody to be determined in amethod according to the current invention is an antibody that binds tohuman A-beta and human transferrin receptor. This antibody is abispecific antibody consisting of a full-length core antibody and afused Fab fragment in which certain domains are crosswise exchanged.Thus, the resulting bispecific antibody is asymmetric. Therefore, thebispecific antibodies are produced using the heterodimerizationtechnology called knobs-into-holes using a first heavy chain with theso-called knob mutations (HCknob) and a second heavy chain with theso-called hole mutations (HChole).

Exemplary antibody 0012 is composed of four polypeptides that have theamino acid sequence of SEQ ID NO: 04 to 07.

Exemplary antibody 0015 is composed of four polypeptides that have theamino acid sequence of SEQ ID NO: 08 to 11.

Exemplary antibody 0020 is composed of three polypeptides that have theamino acid sequence of SEQ ID NO: 12 to 14.

Exemplary antibody 0024 is composed of four polypeptides that have theamino acid sequence of SEQ ID NO: 15 to 18.

In one aspect, the therapeutic antibody is a bispecific antibodycomprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other, and    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor.

In another embodiment, the therapeutic antibody is a bispecific antibodycomprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other,    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor,    -   wherein the human A-beta binding site comprises a heavy chain        variable domain (VH) sequence having at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to        the amino acid sequence of SEQ ID NO: 19 and a light chain        variable domain (VL) sequence having at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to        the amino acid sequence of SEQ ID NO: 20, and    -   wherein the human transferrin receptor binding site comprises a        heavy chain variable domain (VH) sequence having at least 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence        identity to the amino acid sequence of SEQ ID NO: 21 and a light        chain variable domain (VL) sequence having at least 90%, 91%,        92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence        identity to the amino acid sequence of SEQ ID NO: 22.

In certain embodiments, a VH sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but a binding site comprising that sequenceretains the ability to bind to its antigen. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 19 or 21. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs).

In certain embodiments, a VL sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but a binding site comprising that sequenceretains the ability to bind to its antigen. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 20 or 22. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs).

In one embodiment, the human A-beta binding site comprises the VHsequence as in SEQ ID NO: 19, including post-translational modificationsof that sequence and the VL sequence as in SEQ ID NO: 20.

In one embodiment, the human transferrin receptor-binding site comprisesthe VH sequence as in SEQ ID NO: 21, including post-translationalmodifications of that sequence and the VL sequence as in SEQ ID NO: 22.

In one embodiment, the bispecific antibody comprises

-   -   i) a light chain that has a sequence identity to SEQ ID NO: 23        of at least 70%, at least 80%, at least 90%, or 95% or more,    -   ii) a heavy chain that has a sequence identity to SEQ ID NO: 24        of at least 70%, at least 80%, at least 90%, or 95% or more,    -   iii) a light chain that has a sequence identity to SEQ ID NO: 25        of at least 70%, at least 80%, at least 90%, or 95% or more, and    -   iv) a heavy chain Fab fragment that has a sequence identity to        SEQ ID NO: 26 of at least 70%, at least 80%, at least 90%, or        95% or more,    -   wherein

SEQ ID NO: 23 has the amino acid sequenceDIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC,SEQ ID NO: 24 has the amino acid sequenceQVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,SEQ ID NO: 25 has the amino acid sequenceAIQLTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYASSNVDNTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSC,and SEQ ID NO: 26 has the amino acid sequenceQSMQESGPGLVKPSQTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISKTSTTVSLKLSSVTAADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In another embodiment, the therapeutic antibody is a bispecific antibodycomprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other,    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor,    -   wherein the human A-beta binding site comprises a heavy chain        variable domain (VH) that has the amino acid sequence of SEQ ID        NO: 19 and a light chain variable domain (VL) that has the amino        acid sequence of SEQ ID NO: 20, and    -   wherein the human transferrin receptor-binding site comprises a        heavy chain variable domain (VH) that has the amino acid        sequence of SEQ ID NO: 21 and a light chain variable domain (VL)        that has the amino acid sequence of SEQ ID NO: 22.

In another embodiment, the therapeutic antibody is a bispecific antibodycomprising

-   -   a) one full length antibody comprising two pairs each of a full        length antibody light chain and a full length antibody heavy        chain, wherein the binding sites formed by each of the pairs of        the full length heavy chain and the full length light chain        specifically bind to a first antigen, wherein the full length        antibody comprises an Fc-region that is formed by the Fc-region        polypeptides, each comprising the CH1, CH2 and CH3 domain, of        the two full length heavy chains, and    -   b) one additional Fab fragment, wherein the additional Fab        fragment is fused to the C-terminus of one heavy chain of the        full length antibody, wherein the binding site of the additional        Fab fragment specifically binds to a second antigen,    -   wherein each of the full length antibody light chains comprises        in the constant light chain domain (CL) at position 123 the        amino acid residue arginine (instead of the wild-type glutamic        acid residue; E123R mutation) and at position 124 the amino acid        residue lysine (instead of the wild-type glutamine residue;        Q124K mutation) (numbering according to Kabat),    -   wherein each of the full length antibody heavy chains comprises        in the first constant heavy chain domain (CH1) at position 147        an glutamic acid residue (instead of the wild-type lysine        residue; K147E mutation) and at position 213 an glutamic acid        residue (instead of the wild-type lysine amino acid residue;        K213E mutation) (numbering according to Kabat EU index),    -   wherein the additional Fab fragment specifically binding to the        second antigen comprises a domain crossover such that the        constant light chain domain (CL) and the constant heavy chain        domain 1 (CH1) are replaced by each other,    -   wherein the first antigen is human A-beta protein and the second        antigen is human transferrin receptor,    -   wherein the human A-beta binding site comprises a heavy chain        variable domain (VH) that has the amino acid sequence of SEQ ID        NO: 19 and a light chain variable domain (VL) that has the amino        acid sequence of SEQ ID NO: 20,    -   wherein the human transferrin receptor binding site comprises a        heavy chain variable domain (VH) that has the amino acid        sequence of SEQ ID NO: 21 and a light chain variable domain (VL)        that has the amino acid sequence of SEQ ID NO: 22, and    -   wherein the Fc-region polypeptides are    -   a) of the human subclass IgG1,    -   b) of the human subclass IgG4,    -   c) of the human subclass IgG1 with the mutations L234A, L235A        and P329G,    -   d) of the human subclass IgG4 with the mutations S228P, L235E        and P329G,    -   e) of the human subclass IgG1 with the mutations L234A, L235A        and P329G in both Fc-region polypeptides and the mutations T366W        and S354C in one Fc-region polypeptide and the mutations T366S,        L368A, Y407V and Y349C in the respective other Fc-region        polypeptide,    -   f) of the human subclass IgG4 with the mutations S228P and P329G        in both Fc-region polypeptides and the mutations T366W and S354C        in one Fc-region polypeptide and the mutations T366S, L368A,        Y407V and Y349C in the respective other Fc-region polypeptide,    -   g) of the human subclass IgG1 with the mutations L234A, L235A,        P329G, I253A, H310A and H435A in both Fc-region polypeptides and        the mutations T366W and S354C in one Fc-region polypeptide and        the mutations T366S, L368A, Y407V and Y349C in the respective        other Fc-region polypeptide, or    -   h) of the human subclass IgG1 with the mutations L234A, L235A,        P329G, M252Y, S254T and T256E in both Fc-region polypeptides and        the mutations T366W and S354C in one Fc-region polypeptide and        the mutations T366S, L368A, Y407V and Y349C in the respective        other Fc-region polypeptide.

B. Exemplary Anti-Transferrin Receptor Antibodies

The anti-transferrin receptor binding site of a therapeutic antibody tobe determined in a method according to the current invention have anoff-rate for binding to the human transferrin receptor that is within acertain range in order to ensure proper BBB shuttling. This range isdefined at the one end by the off-rate of the murine anti-transferrinreceptor antibody 128.1 (variable domain amino acid sequences given inSEQ ID NO: 27 and 28) determined by surface plasmon resonance for thecynomolgus transferrin receptor and at the other end by 5% of thatoff-rate (i.e. a 20-times slower dissociation). The off-rate for thehuman transferrin receptor should be between and including 0.1 l/s and0.005 l/s.

One aspect as reported herein is an anti-transferrin receptor antibodythat specifically binds to human transferrin receptor and cynomolgustransferrin receptor, which comprises

-   -   i) a humanized heavy chain variable domain derived from the        heavy chain variable domain of SEQ ID NO: 29, and    -   ii) a humanized light chain variable domain derived from the        light chain variable domain of SEQ ID NO: 30,    -   wherein the antibody has an off-rate for the human transferrin        receptor that is equal to or less than (i.e. at most) the        off-rate of the anti-transferrin receptor antibody 128.1 for the        cynomolgus transferrin receptor,    -   whereby the off-rates are determined by surface plasmon        resonance, and    -   whereby the anti-transferrin receptor antibody 128.1 has a heavy        chain variable domain of SEQ ID NO: 27 and a light chain        variable domain of SEQ ID NO: 28.

In one embodiment, the off-rate for the human transferrin receptor isbetween and including 0.1 l/s and 0.005 l/s.

In one embodiment, the antibody has in the light chain variable domainat position 80 a proline amino acid residue (P) (numbering according toKabat).

In one embodiment, the antibody has in the light chain variable domainat position 91 an asparagine amino acid residue (N) (numbering accordingto Kabat).

In one embodiment, the antibody has in the light chain variable domainat position 93 an alanine amino acid residue (A) (numbering according toKabat).

In one embodiment, the antibody has in the heavy chain variable domainat position 100 g a serine amino acid residue (S) (numbering accordingto Kabat).

In one embodiment, the antibody has in the heavy chain variable domainat position 100 g a glutamine amino acid residue (Q) (numberingaccording to Kabat).

In one embodiment, the antibody has in the heavy chain variable domainat position 65 a serine amino acid residue (S) (numbering according toKabat).

In one embodiment, the antibody has in the heavy chain variable domainat position 105 a glutamine amino acid residue (Q) (numbering accordingto Kabat).

In one embodiment, the antibody the antibody has in the light chainvariable domain at position 80 a proline amino acid residue (P), in thelight chain variable domain at position 91 an asparagine amino acidresidue (N), in the light chain variable domain at position 93 analanine amino acid residue (A), in the heavy chain variable domain atposition 100 g a serine amino acid residue (S), in the heavy chainvariable domain at position 65 a serine amino acid residue (S), and inthe heavy chain variable domain at position 105 a glutamine amino acidresidue (Q) (numbering according to Kabat).

In one embodiment, the antibody the antibody has in the light chainvariable domain at position 80 a proline amino acid residue (P), in thelight chain variable domain at position 91 an asparagine amino acidresidue (N), in the light chain variable domain at position 93 analanine amino acid residue (A), in the heavy chain variable domain atposition 100 g a glutamine amino acid residue (Q), in the heavy chainvariable domain at position 65 a serine amino acid residue (S), and inthe heavy chain variable domain at position 105 a glutamine amino acidresidue (Q) (numbering according to Kabat).

Such anti-transferrin receptor bispecific antibodies can be used asblood-brain-barrier shuttle module to deliver a brain effector entityacross the blood-brain-barrier into the brain. The blood-brain-barriershuttle module is a monovalent binding entity that specifically binds tothe human transferrin receptor. The anti-transferrin receptor bispecificantibodies when used as blood-brain-barrier shuttle module are useful,e.g., for the diagnosis or treatment of neurological disorders, such asAlzheimer's disease, Parkinson's Disease and Alzheimer's Disease withParkinson's Disease co-morbidity.

In one embodiment, the anti-transferrin receptor binding site of thetherapeutic antibody comprises the heavy chain variable domain of SEQ IDNO: 31 and the light chain variable domain of SEQ ID NO: 32 whichreflect with respect to the human transferrin receptor the bindingproperties of the murine antibody 128.1 with respect to the cynomolgustransferrin receptor regarding the binding off-rate.

In one embodiment, the anti-transferrin receptor binding site of thetherapeutic antibody specifically binds to human transferrin receptor(huTfR) and cynomolgus transferrin receptor (cyTfR) and comprises i) ahumanized heavy chain variable domain derived from the heavy chainvariable domain of SEQ ID NO: 29 and ii) a humanized light chainvariable domain derived from the light chain variable domain of SEQ IDNO: 30, wherein the light chain variable domain has at position 80 aproline amino acid residue (P), at position 91 an asparagine amino acidresidue (N) and at position 93 an alanine amino acid residue (A)(numbering according to Kabat).

In one embodiment, the anti-transferrin receptor binding site of thetherapeutic antibody further has in the heavy chain variable domain atposition 100 g a serine amino acid residue (S) (numbering according toKabat).

In one embodiment, the anti-transferrin receptor binding site of thetherapeutic antibody further has in the heavy chain variable domain atposition 65 a serine amino acid residue (S) (numbering according toKabat).

In one embodiment, the anti-transferrin receptor binding site of thetherapeutic antibody further has in the heavy chain variable domain atposition 105 a glutamine amino acid residue (Q) (numbering according toKabat).

In one embodiment, the anti-transferrin receptor binding site of thetherapeutic antibody specifically binds to human transferrin receptor(huTfR) and cynomolgus transferrin receptor (cyTfR) and comprises i) ahumanized heavy chain variable domain derived from the heavy chainvariable domain of SEQ ID NO: 29 and ii) a humanized light chainvariable domain derived from the light chain variable domain of SEQ IDNO: 30, wherein the therapeutic antibody has an off-rate in the unit 1/sfor the human transferrin receptor that is equal to or less than (i.e.at most) the off-rate in the unit 1/s of the anti-transferrin receptorantibody 128.1 for the cynomolgus transferrin receptor, whereby theoff-rates are determined by surface plasmon resonance, and whereby theanti-transferrin receptor antibody 128.1 has a heavy chain variabledomain of SEQ ID NO: 27 and a light chain variable domain of SEQ ID NO:28.

In one embodiment, the anti-transferrin receptor binding site of thetherapeutic antibody has an off-rate in the unit 1/s for the humantransferrin receptor that is i) equal to or less than (i.e. at most) theoff-rate in the unit 1/s of the anti-transferrin receptor antibody 128.1for the cynomolgus transferrin receptor and ii) equal to or more than(i.e. at least) 5% of the off-rate in the unit 1/s of theanti-transferrin receptor antibody 128.1 for the cynomolgus transferrinreceptor.

In one embodiment, the anti-transferrin receptor binding site of thetherapeutic antibody comprises (a) a HVR-H1 comprising the amino acidsequence of SEQ ID NO: 33; (b) a HVR-H2 comprising the amino acidsequence of SEQ ID NO: 34; (c) a HVR-H3 comprising the amino acidsequence of SEQ ID NO: 35, 36 or 37, in one preferred embodiment, SEQ IDNO: 36; (d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO:38; (e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39;and (f) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

In any of the above embodiments, an anti-transferrin receptor-bindingsite is humanized. In one embodiment, an anti-transferrinreceptor-binding site comprises HVRs as in any of the above embodiments,and further comprises an acceptor human framework, e.g. a humanimmunoglobulin framework or a human consensus framework.

In one embodiment, the antibody is a bispecific antibody comprising atleast one pair of a heavy chain variable domain of SEQ ID NO: 31 and alight chain variable domain of SEQ ID NO: 32 forming a binding site forthe transferrin receptor and at least one pair of a heavy chain variabledomain of SEQ ID NO: 41 and a light chain variable domain of SEQ ID NO:42 binding site for human CD20. In one embodiment, the heavy chainvariable region comprises a replacement of the amino acid residue atKabat position 11 with any amino acid but leucine. In one embodiment,the substitution comprises a replacement of the amino acid residue atKabat position 11 with a nonpolar amino acid. In one preferredembodiment, the substitution comprises a replacement of the amino acidresidue at Kabat position 11 in the heavy chain variable domain of SEQID NO: 41 with an amino acid residue selected from the group consistingof valine, leucine, isoleucine, serine, and phenylalanine.

In one embodiment, the antibody is a bispecific antibody comprising atleast one pair of a heavy chain variable domain of SEQ ID NO: 31 and alight chain variable domain of SEQ ID NO: 32 forming a binding site forthe transferrin receptor and at least one pair of a heavy chain variabledomain of SEQ ID NO: 43 and a light chain variable domain of SEQ ID NO:44 binding site for human alpha-synuclein.

In one embodiment, the antibody is a bispecific antibody comprising atleast one pair of a heavy chain variable domain of SEQ ID NO: 31 and alight chain variable domain of SEQ ID NO: 32 forming a binding site forthe transferrin receptor and at least one pair of a humanized heavychain variable domain derived from SEQ ID NO: 45 and a humanized lightchain variable domain derived from SEQ ID NO: 46 binding site for humanalpha-synuclein.

In one embodiment, the antibody is a bispecific antibody comprising atleast one pair of a heavy chain variable domain of SEQ ID NO: 31 and alight chain variable domain of SEQ ID NO: 32 forming a binding site forthe transferrin receptor and at least one pair of a humanized heavychain variable domain derived from SEQ ID NO: 47 and a humanized lightchain variable domain derived from SEQ ID NO: 48 binding site for humanalpha-synuclein.

In one embodiment, the antibody is a bispecific antibody comprising atleast one pair of a heavy chain variable domain of SEQ ID NO: 31 and alight chain variable domain of SEQ ID NO: 32 forming a binding site forthe transferrin receptor and at least one pair of a humanized heavychain variable domain derived from SEQ ID NO: 49 and a humanized lightchain variable domain derived from SEQ ID NO: 50 binding site for humanalpha-synuclein.

In one embodiment, the antibody is a bispecific antibody comprising atleast one pair of a heavy chain variable domain of SEQ ID NO: 31 and alight chain variable domain of SEQ ID NO: 32 forming a binding site forthe transferrin receptor and at least one pair of a humanized heavychain variable domain derived from SEQ ID NO: 51 and a humanized lightchain variable domain derived from SEQ ID NO: 52 binding site for humanalpha-synuclein.

In one embodiment, the antibody is a bispecific antibody comprising atleast one pair of a heavy chain variable domain of SEQ ID NO: 31 and alight chain variable domain of SEQ ID NO: 32 forming a binding site forthe transferrin receptor and at least one pair of a humanized heavychain variable domain derived from SEQ ID NO: 53 and a humanized lightchain variable domain derived from SEQ ID NO: 54 binding site for humanalpha-synuclein.

In one embodiment, the antibody is a bispecific antibody comprising atleast one pair of a heavy chain variable domain of SEQ ID NO: 31 and alight chain variable domain of SEQ ID NO: 32 forming a binding site forthe transferrin receptor and a binding site for i) glucocerebrosidasethat has the amino acid sequence of SEQ ID NO: 55, or ii) a functionalvariant of SEQ ID NO: 55 having at least 70% sequence identity, or iii)a functional variant of SEQ ID NO: 55 having one or more amino acidmutations, deletions or insertions, or iv) a truncated functionalvariant of SEQ ID NO: 55 having at least one amino acid residue at theN-terminus or the C-terminus or within the amino acid sequence deleted,or v) a combination of iii) and iv).

In another embodiment, the therapeutic antibody is a multispecificantibody. In one such embodiment, the multispecific antibody comprises afirst antigen-binding site, which binds TfR, and a secondantigen-binding site, which binds a brain antigen. In one such aspect,the brain antigen is selected from the group consisting of:beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor(EGFR), human epidermal growth factor receptor 2 (HER2), tau,apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR),glucocerebrosidase, and caspase 6. In another embodiment, themultispecific antibody binds both TfR and BACE1. In another embodiment,the multispecific antibody binds both TfR and Abeta. In anotherembodiment, the multispecific antibody binds both TfR and alphasynuclein. In another embodiment, the multispecific antibody binds bothTfR and CD20. In another embodiment, the multispecific antibody bindsboth TfR and glucocerebrosidase. In another embodiment, the therapeuticcompound is a neurological disorder therapeutic antibody.

In one embodiment, the effector function is reduced or eliminated by atleast one modification of the Fc region. In one embodiment, the effectorfunction or complement activation function is reduced or eliminated bydeletion of all or a portion of the Fc region, or by engineering theantibody such that it does not include an Fc region or non-Fc regioncompetent for effector function or complement activation function. Inone embodiment, the at least one modification of the Fc region isselected from: a point mutation of the Fc region to impair binding toone or more Fc receptors selected from the following positions: 238,239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293,294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 30 335, 338,340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, and439; a point mutation of the Fc region to impair binding to C1q selectedfrom the following positions: 270, 322, 329, and 321; eliminating someor all of the Fc region, and a point mutation at position 132 of the CH1domain. In one embodiment, the modification is a point mutation of theFc region to impair binding to C1q selected from the followingpositions: 270, 322, 329, and 321. In another embodiment, themodification is elimination of some or all of the Fc region. In anotherembodiment, complement-triggering function is reduced or eliminated bydeletion of all or a portion of the Fc region, or by engineering theantibody such that it does not include an Fc region that engages thecomplement pathway. In one embodiment, the antibody is selected from aFab or a single chain antibody. In another embodiment, the non-Fc regionof the antibody is modified to reduce or eliminate activation of thecomplement pathway by the antibody. In one embodiment, the modificationis a point mutation of the CH1 region to impair binding to C3. In oneembodiment, the point mutation is at position 132 (see, e.g., Vidarte etal., J. Biol. Chem. 276 (2001) 38217-38223).

In one aspect of the above embodiment, the affinity of the antibody forTfR is decreased, as measured relative to a wild-type antibody of thesame isotype not having lowered affinity for TfR. In one such aspect,the antibody has a K_(D) or IC₅₀ for TfR of about 1 pM to about 100 μM.

In one embodiment, the antibody as reported herein is effector functionsilent. In one embodiment, the antibody has no effector function. In oneembodiment, the antibody is of the human IgG1 subclass and has themutations L234A, L235A and P329G in both heavy chains (numberingaccording to the EU index of Kabat).

In one embodiment, the antibody is

-   -   a) a full length antibody of the human subclass IgG1, or    -   b) a full length antibody of the human subclass IgG4, or    -   c) a full-length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G,    -   d) a full-length antibody of the human subclass IgG4 with the        mutations S228P, L235E and optionally P329G,    -   e) a full length antibody of the human subclass IgG1 with the        mutations L234A, L235A and P329G in both heavy chains and the        mutations T366W and S354C in one heavy chain and the mutations        T366S, L368A, Y407V and Y349C in the respective other heavy        chain, or    -   f) a full-length antibody of the human subclass IgG4 with the        mutations S228P and optionally P329G in both heavy chains and        the mutations T366W and S354C in one heavy chain and the        mutations T366S, L368A, Y407V and Y349C in the respective other        heavy chain.

In one embodiment, the bispecific therapeutic antibody comprises

-   -   i) a homodimeric Fc-region of the human IgG1 subclass optionally        with the mutations P329G, L234A and L235A, or    -   ii) a homodimeric Fc-region of the human IgG4 subclass        optionally with the mutations P329G, S228P and L235E, or    -   iii) a heterodimeric Fc-region whereof        -   a) one Fc-region polypeptide comprises the mutation T366W,            and the other Fc-region polypeptide comprises the mutations            T366S, L368A and Y407V, or        -   b) one Fc-region polypeptide comprises the mutations T366W            and Y349C, and the other Fc-region polypeptide comprises the            mutations T366S, L368A, Y407V, and S354C, or        -   c) one Fc-region polypeptide comprises the mutations T366W            and S354C, and the other Fc-region polypeptide comprises the            mutations T366S, L368A, Y407V and Y349C,        -   or    -   iv) a heterodimeric Fc-region of the human IgG4 subclass whereof        both Fc-region polypeptides comprise the mutations P329G, L234A        and L235A and        -   a) one Fc-region polypeptide comprises the mutation T366W,            and the other Fc-region polypeptide comprises the mutations            T366S, L368A and Y407V, or        -   b) one Fc-region polypeptide comprises the mutations T366W            and Y349C, and the other Fc-region polypeptide comprises the            mutations T366S, L368A, Y407V, and S354C, or        -   c) one Fc-region polypeptide comprises the mutations T366W            and S354C, and the other Fc-region polypeptide comprises the            mutations T366S, L368A, Y407V and Y349C,        -   or    -   v) a heterodimeric Fc-region of the human IgG4 subclass whereof        both Fc-region polypeptides comprise the mutations P329G, S228P        and L235E and        -   a) one Fc-region polypeptide comprises the mutation T366W,            and the other Fc-region polypeptide comprises the mutations            T366S, L368A and Y407V, or        -   b) one Fc-region polypeptide comprises the mutations T366W            and Y349C, and the other Fc-region polypeptide comprises the            mutations T366S, L368A, Y407V, and S354C, or        -   c) one Fc-region polypeptide comprises the mutations T366W            and S354C, and the other Fc-region polypeptide comprises the            mutations T366S, L368A, Y407V and Y349C.

Immunoassays

The principles of different immunoassays are described in the art. Forexample, Hage, D. S. (Anal. Chem. 71 (1999) 294R-304R). Lu, B., et al.(Analyst 121 (1996) 29R-32R) report the orientated immobilization ofantibodies for the use in immunoassays. Avidin-biotin-mediatedimmunoassays are reported, for example, by Wilchek, M., and Bayer, E.A., in Methods Enzymol. 184 (1990) 467-469.

Monoclonal antibodies and their constant domains contain a number ofreactive amino acid side chains for conjugating to a member of a bindingpair, such as a polypeptide/protein, a polymer (e.g. PEG, cellulose orpolystyrol), or an enzyme. Chemical reactive groups of amino acids are,for example, amino groups (lysins, alpha-amino groups), thiol groups(cystins, cysteines, and methionins), carboxylic acid groups (asparticacids, glutamic acids), and sugar-alcoholic groups. Such methods aree.g. described by Aslam M., and Dent, A., in “Bioconjugation”, MacMillanRef. Ltd. 1999, pages 50-100.

One of the most common reactive groups of antibodies is the aliphaticε-amine of the amino acid lysine. In general, nearly all antibodiescontain abundant lysine. Lysine amines are reasonably good nucleophilesabove pH 8.0 (pKa=9.18) and therefore react easily and cleanly with avariety of reagents to form stable bonds. Amine-reactive reagents reactprimarily with lysins and the α-amino groups of proteins. Reactiveesters, particularly N-hydroxy-succinimide (NETS) esters, are among themost commonly employed reagents for modification of amine groups. Theoptimum pH for reaction in an aqueous environment is pH 8.0 to 9.0.Isothiocyanates are amine-modification reagents and form thiourea bondswith proteins. They react with protein amines in aqueous solution(optimally at pH 9.0 to 9.5). Aldehydes react under mild aqueousconditions with aliphatic and aromatic amines, hydrazines, andhydrazides to form an imine intermediate (Schiff s base). A Schiff sbase can be selectively reduced with mild or strong reducing agents(such as sodium borohydride or sodium cyanoborohydride) to derive astable alkyl amine bond. Other reagents that have been used to modifyamines are acid anhydrides. For example, diethylenetriaminepentaaceticanhydride (DTPA) is a bifunctional chelating agent that contains twoamine-reactive anhydride groups. It can react with N-terminal andε-amine groups of amino acids to form amide linkages. The anhydriderings open to create multivalent, metal-chelating arms able to bindtightly to metals in a coordination complex.

Another common reactive group in antibodies is the thiol residue fromthe sulfur-containing amino acid cystine and its reduction productcysteine (or half cystine). Cysteine contains a free thiol group, whichis more nucleophilic than amines and is generally the most reactivefunctional group in a protein. Thiols are generally reactive at neutralpH, and therefore can be coupled to other molecules selectively in thepresence of amines. Since free sulfhydryl groups are relativelyreactive, proteins with these groups often exist with them in theiroxidized form as disulfide groups or disulfide bonds. In such proteins,reduction of the disulfide bonds with a reagent such as dithiotreitol(DTT) is required to generate the reactive free thiol. Thiol-reactivereagents are those that will couple to thiol groups on polypeptides,forming thioether-coupled products. These reagents react rapidly atslight acidic to neutral pH and therefore can be reacted selectively inthe presence of amine groups. The literature reports the use of severalthiolating crosslinking reagents such as Traut's reagent(2-iminothiolane), succinimidyl (acetylthio) acetate (SATA), andsulfosuccinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate(Sulfo-LC-SPDP) to provide efficient ways of introducing multiplesulfhydryl groups via reactive amino groups. Haloacetyl derivatives,e.g. iodoacetamides, form thioether bonds and are reagents for thiolmodification. Further useful reagents are maleimides. The reaction ofmaleimides with thiol-reactive reagents is essentially the same as withiodoacetamides. Maleimides react rapidly at slight acidic to neutral pH.

Another common reactive group in antibodies are carboxylic acids.Antibodies contain carboxylic acid groups at the C-terminal position andwithin the side chains of aspartic acid and glutamic acid. Therelatively low reactivity of carboxylic acids in water usually makes itdifficult to use these groups to selectively modify polypeptides andantibodies. When this is done, the carboxylic acid group is usuallyconverted to a reactive ester by the use of a water-soluble carbodiimideand reacted with a nucleophilic reagent such as an amine, hydrazide, orhydrazine. The amine-containing reagent should be weakly basic in orderto react selectively with the activated carboxylic acid in the presenceof the more highly basic ε-amines of lysine to form a stable amide bond.Protein crosslinking can occur when the pH is raised above 8.0.

Sodium periodate can be used to oxidize the alcohol part of a sugarwithin a carbohydrate moiety attached to an antibody to an aldehyde.Each aldehyde group can be reacted with an amine, hydrazide, orhydrazine as described for carboxylic acids. Since the carbohydratemoiety is predominantly found on the crystallizable fragment region(Fc-region) of an antibody, conjugation can be achieved throughsite-directed modification of the carbohydrate away from theantigen-binding site. A Schiff s base intermediate is formed, which canbe reduced to an alkyl amine through the reduction of the intermediatewith sodium cyanoborohydride (mild and selective) or sodium borohydride(strong) water-soluble reducing agents.

The conjugation of a tracer and/or capture and/or detection antibody toits conjugation partner can be performed by different methods, such aschemical binding, or binding via a binding pair. The term “conjugationpartner” as used herein denotes e.g. solid supports, polypeptides,detectable labels, members of specific binding pairs. In one embodiment,the conjugation of the capture and/or tracer and/or detection antibodyto its conjugation partner is performed by chemically binding viaN-terminal and/or ε-amino groups (lysine), ε-amino groups of differentlysins, carboxy-, sulfhydryl-, hydroxyl-, and/or phenolic functionalgroups of the amino acid backbone of the antibody, and/or sugar alcoholgroups of the carbohydrate structure of the antibody. In one embodiment,the capture antibody is conjugated to its conjugation partner via abinding pair. In one preferred embodiment, the capture antibody isconjugated to biotin and immobilization to a solid support is performedvia solid support immobilized avidin or streptavidin. In one embodiment,the capture antibody is conjugated to its conjugation partner via abinding pair. In one preferred embodiment, the tracer antibody isconjugated to digoxygenin by a covalent bond as detectable label.

Chromogens (fluorescent or luminescent groups and dyes), enzymes,NMR-active groups or metal particles, haptens, e.g. digoxygenin, areexamples of “detectable labels”. The detectable label can also be aphotoactivatable crosslinking group, e.g. an azido or an azirine group.Metal chelates, which can be detected by electrochemiluminescense, arealso preferred signal-emitting groups, with particular preference beinggiven to ruthenium chelates, e.g. a ruthenium (bispyridyl)32+chelate.Suitable ruthenium labeling groups are described, for example, in EP 0580 979, WO 90/05301, WO 90/11511, and WO 92/14138. For directdetection, the labeling group can be selected from any known detectablemarker groups, such as dyes, luminescent labeling groups such aschemiluminescent groups, e.g. acridinium esters or dioxetanes, orfluorescent dyes, e.g. fluorescein, coumarin, rhodamine, oxazine,resorufin, cyanine and derivatives thereof. Other examples of labelinggroups are luminescent metal complexes, such as ruthenium or europiumcomplexes, enzymes, e.g. as used for ELISA or for CEDIA (Cloned EnzymeDonor Immunoassay, e.g. EP-A-0 061 888), and radioisotopes.

Indirect detection systems comprise, for example, that the detectionreagent, e.g., the detection antibody is labeled with a first partner ofa binding pair. Examples of suitable binding pairs are antigen/antibody,biotin or biotin analogues such as aminobiotin, iminobiotin ordesthiobiotin/avidin or Streptavidin, sugar/lectin, nucleic acid ornucleic acid analogue/complementary nucleic acid, and receptor/ligand,e.g., steroid hormone receptor/steroid hormone. In one preferredembodiment, the first binding pair members comprise hapten, antigen andhormone. In one preferred embodiment, the hapten is selected from thegroup consisting of digoxin, digoxygenin and biotin and analoguesthereof. The second partner of such binding pair, e.g. an antibody,Streptavidin, etc., usually is labeled to allow for direct detection,e.g., by the labels as mentioned above.

Immunoassays can be performed generally in three different formats. Oneis with direct detection, one with indirect detection, or by a sandwichassay. The direct detection immunoassay uses a detection (or tracer)antibody that can be measured directly. An enzyme or other moleculeallows for the generation of a signal that will produce a color,fluorescence, or luminescence that allow for the signal to be visualizedor measured (radioisotopes can also be used, although it is not commonlyused today). In an indirect assay a primary antibody that binds to theanalyte is used to provide a defined target for a secondary antibody(tracer antibody) that specifically binds to the target provided by theprimary antibody (referred to as detector or tracer antibody). Thesecondary antibody generates the measurable signal. The sandwich assaymakes use of two antibodies, a capture and a tracer (detector) antibody.The capture antibody is used to bind (immobilize) analyte from solutionor bind to it in solution. This allows the analyte to be specificallyremoved from the sample. The tracer (detector) antibody is used in asecond step to generate a signal (either directly or indirectly asdescribed above). The sandwich format requires two antibodies each witha distinct epitope on the target molecule. In addition, they must notinterfere with one another, as both antibodies must be bound to thetarget at the same time.

Different principles for the determination of bispecific antibodies inan immunoassay are known to a person skilled in the art:

-   -   1) capture using        -   one of the antigens;        -   an anti-idiotypic antibody against one of the binding sites;    -   2) detection using        -   the respective other antigen;        -   an anti-idiotypic antibody against the respective other            binding site;

These can be combined independently of each other.

Blood-Brain-Barrier Penetrating Antibodies of the Method According tothe Invention

The present invention relates in one aspect to the determination of theconcentration of a bispecific antibody for use in the treatment of adisease in a patient in brain tissue,

-   -   wherein the bispecific therapeutic antibody comprises        -   i) an (effector function competent) Fc-region,        -   ii) two binding sites specifically binding to a first (cell            surface) target, and        -   iii) one binding site specifically binding to a second (cell            surface) target,    -   wherein the treatment has reduced side effect after        administration,    -   wherein the side effect is one or more selected from the group        consisting of vasodilation, bronchoconstriction, laryngeal        edema, drop of cardiac pressure, and hypothermia.

In one embodiment, the two binding sites specifically binding to thefirst target and the binding site specifically binding to the secondtarget are arranged in opposite directions, i.e. one is conjugated tothe N-terminus of the Fc-region and the other is conjugated to theC-terminus of the Fc-region.

In one embodiment, the first (cell surface) target and the second (cellsurface) target are different.

In one embodiment, the binding sites specifically binding to the first(cell surface) target and the binding site specifically binding to thesecond (cell surface) target are located at opposite ends (i.e. thosespecifically binding to the first target are both/each at an N-terminalend of a (full length) antibody heavy chain and that to the secondtarget is at the C-terminal end of one of the (full length) antibodyheavy chains of the bispecific antibody.

In one embodiment, the binding sites specifically binding to the first(cell surface) target and the binding site specifically binding to thesecond (cell surface) target are located at opposite ends of thebispecific antibody, i.e. one of the binding sites specifically bindingto the first target is conjugated to the first N-terminus of theFc-region and the other is conjugated to the second N-terminus of theFc-region and the binding site that specifically binds to the secondtarget is conjugated to one of the C-termini of the Fc-region.

In one embodiment, the binding site specifically binding to the second(cell surface) target is linked to one of the binding sites specificallybinding to the first (cell surface) target by a peptidic linker. In oneembodiment, the peptidic linker has the amino acid sequence of SEQ IDNO: 56 or 57.

In one embodiment, the binding site specifically binding to a second(cell surface) target is within the Fc-region, wherein at least onestructural loop region of any of a CH2 domain, a CH3 domain, or a CH4domain comprises at least one modification enabling the binding of saidat least one modified loop region to the second (cell surface) targetwherein the unmodified immunoglobulin constant domain does not bind tosaid target.

In one embodiment, the binding sites are pairs of an antibody heavychain variable domain and an antibody light chain variable domain.

In one embodiment, the bispecific therapeutic antibody comprises

-   -   i) a pair of a first antibody light chain and a first antibody        heavy chain,    -   ii) a pair of a second antibody light chain and a second        antibody heavy chain, and    -   iii) an additional antibody fragment selected from the group        consisting of scFv, Fab, scFab, dAb fragment, DutaFab and        CrossFab,    -   wherein the pair of antibody chains of i) and ii) comprise the        binding sites specifically binding to the first (cell surface)        target and the additional antibody fragment of iii) comprises        the binding site specifically binding to the second (cell        surface) target.

In one embodiment, the additional antibody fragment of iii) isconjugated either directly or via a peptidic linker either to the firstantibody heavy chain or to the second antibody heavy chain. In oneembodiment, the additional antibody fragment of iii) is conjugatedeither directly or via a peptidic linker to the C-terminus of theantibody heavy chain of i) or ii). In one embodiment, the peptidiclinker has the amino acid sequence of SEQ ID NO: 56 or 57. In oneembodiment, the first antibody light chain and the second antibody lightchain have the same amino acid sequence and the first antibody heavychain and the second antibody heavy chain differ by mutations requiredfor heterodimerization. In one embodiment, the mutations required forheterodimerization are the knobs-into-hole mutations. In one embodiment,the antibody heavy chain not conjugated to the additional antibodyfragment of iii) does not comprise i) the C-terminal lysine residue orii) the C-terminal glycine-lysine dipeptide.

In one embodiment, the first target is a brain target and the secondtarget is the human transferrin receptor. In one embodiment, the firsttarget is a brain target and the second target is the human transferrinreceptor 1.

In one embodiment, the brain target is selected from the groupconsisting of beta-secretase 1 (BACE1), human amyloid beta (Abeta),epidermal growth factor receptor (EGFR), human epidermal growth factorreceptor 2 (HER2), human Tau protein, phosphorylated human Tau protein,apolipoprotein E4 (ApoE4), human alpha-synuclein, human CD20,huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2),parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor(p75NTR), and caspase 6. In one preferred embodiment, the brain targetis selected from the group consisting of human CD20, human Tau protein,phosphorylated human Tau protein, human alpha-synuclein and humanamyloid beta protein. In one preferred embodiment, the brain target ishuman amyloid beta protein. In one embodiment, the brain target isselected from SEQ ID NO: 58, 59, 60, 01, 61.

In one preferred embodiment, the bispecific therapeutic antibodycomprises

-   -   i) a pair of a first antibody light chain and a first antibody        heavy chain comprising a first light chain variable domain and a        first heavy chain variable domain, which form a first binding        site specifically binding to a brain target selected from the        group consisting of human CD20, human Tau protein,        phosphorylated human Tau protein, human alpha-synuclein and        human amyloid beta protein,    -   ii) a pair of a second antibody light chain and a second        antibody heavy chain comprising a second light chain variable        domain and a second heavy chain variable domain, which form a        second binding site specifically binding to the same brain        target as the first binding site,    -   iii) an additional antibody fragment selected from the group        consisting of scFv, Fab, scFab, dAb fragment, DutaFab and        CrossFab, comprising a third light chain variable domain and a        third heavy chain variable domain, which form a third binding        site specifically binding to the human transferrin receptor        (transferrin receptor 1), and    -   iv) a (human) effector function competent Fc-region (of the        human IgG1 subclass),    -   wherein the additional antibody fragment of iii) is conjugated        either directly or via a peptidic linker to the C-terminus of        the antibody heavy chain of i) or ii).

In one embodiment, the additional antibody fragment is a Fab fragment,which specifically bind to a second antigen, and which is fused via apeptidic linker to the C-terminus of one of the heavy chains of i) orii), wherein the constant domains CL and CH1 of the second light chainand the second heavy chain are replaced by each other, comprising athird light chain variable domain and a third heavy chain variabledomain, which form a third binding site specifically binding to thehuman transferrin receptor (transferrin receptor 1).

In one embodiment, the binding site specifically binding to the humantransferrin receptor (transferrin receptor 1) comprises (a) a HVR-H1comprising the amino acid sequence of SEQ ID NO: 33 or 62; (b) a HVR-H2comprising the amino acid sequence of SEQ ID NO: 34 or 63 or 35; (c) aHVR-H3 comprising the amino acid sequence of SEQ ID NO: 36, 37 or 64;(d) a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38 or 65;(e) a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and(f) a HVR-L3 comprising the amino acid sequence of SEQ ID NO: 66 or 40.

In one embodiment, the binding site specifically binding to the humantransferrin receptor (transferrin receptor 1) comprises (a) a HVR-H1comprising the amino acid sequence of SEQ ID NO: 33; (b) a HVR-H2comprising the amino acid sequence of SEQ ID NO: 34; (c) a HVR-H3comprising the amino acid sequence of SEQ ID NO: 37; (d) a HVR-L1comprising the amino acid sequence of SEQ ID NO: 38; (e) a HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (f) a HVR-L3comprising the amino acid sequence of SEQ ID NO: 40.

In one embodiment, the therapeutic antibody comprises one pair of aheavy chain variable domain of SEQ ID NO: 31 and a light chain variabledomain of SEQ ID NO: 32 forming a binding site for the transferrinreceptor (transferrin receptor 1) and at least one (i.e. one or two)pair of a heavy chain variable domain of SEQ ID NO:

19 and a light chain variable domain of SEQ ID NO: 20 (each) forming abinding site for human amyloid beta protein (Abeta).

In one embodiment, the therapeutic antibody comprises one pair of aheavy chain variable domain of SEQ ID NO: 31 and a light chain variabledomain of SEQ ID NO: 32 forming the binding site for the humantransferrin receptor (transferrin receptor 1) and two pairs of a heavychain variable domain of SEQ ID NO: 41 and a light chain variable domainof SEQ ID NO: 42 each forming a binding site for human CD20. In oneembodiment, the heavy chain variable region comprises a replacement ofthe amino acid residue at Kabat position 11 with any amino acid butleucine. In one embodiment, the substitution comprises a replacement ofthe amino acid residue at Kabat position 11 with a nonpolar amino acid.In one preferred embodiment, the substitution comprises a replacement ofthe amino acid residue at Kabat position 11 in the heavy chain variabledomain of SEQ ID NO: 41 with an amino acid residue selected from thegroup consisting of valine, leucine, isoleucine, serine, andphenylalanine.

In one embodiment, the therapeutic antibody comprises one pair of aheavy chain variable domain of SEQ ID NO: 31 and a light chain variabledomain of SEQ ID NO: 32 forming the binding site for the humantransferrin receptor (transferrin receptor 1) and two pairs of a heavychain variable domain of SEQ ID NO: 43 and a light chain variable domainof SEQ ID NO: 44 each forming a binding site for human alpha-synuclein.

In one embodiment, the therapeutic antibody comprises one pair of aheavy chain variable domain of SEQ ID NO: 31 and a light chain variabledomain of SEQ ID NO: 32 forming the binding site for the humantransferrin receptor (transferrin receptor 1) and two pairs of ahumanized heavy chain variable domain derived from SEQ ID NO: 45 and ahumanized light chain variable domain derived from SEQ ID NO: 46 eachforming a binding site for human alpha-synuclein.

In one embodiment, the therapeutic antibody comprises one pair of aheavy chain variable domain of SEQ ID NO: 31 and a light chain variabledomain of SEQ ID NO: 32 forming the binding site for the humantransferrin receptor and two pairs of a humanized heavy chain variabledomain derived from SEQ ID NO: 47 and a humanized light chain variabledomain derived from SEQ ID NO: 48 each forming a binding site for humanalpha-synuclein.

In one embodiment, the therapeutic antibody comprises one pair of aheavy chain variable domain of SEQ ID NO: 31 and a light chain variabledomain of SEQ ID NO: 32 forming the binding site for the humantransferrin receptor (transferrin receptor 1) and two pairs of ahumanized heavy chain variable domain derived from SEQ ID NO: 49 and ahumanized light chain variable domain derived from SEQ ID NO: 50 eachforming a binding site for human alpha-synuclein.

In one embodiment, the therapeutic antibody comprises one pair of aheavy chain variable domain of SEQ ID NO: 31 and a light chain variabledomain of SEQ ID NO: 32 forming the binding site for the humantransferrin receptor (transferrin receptor 1) and two pairs of ahumanized heavy chain variable domain derived from SEQ ID NO: 51 and ahumanized light chain variable domain derived from SEQ ID NO: 52 eachforming a binding site for human alpha-synuclein.

In one embodiment, the therapeutic antibody comprising one pair of aheavy chain variable domain of SEQ ID NO: 31 and a light chain variabledomain of SEQ ID NO: 32 forming the binding site for the humantransferrin receptor (transferrin receptor 1) and two pairs of ahumanized heavy chain variable domain derived from SEQ ID NO: 53 and ahumanized light chain variable domain derived from SEQ ID NO: 54 eachforming a binding site for human alpha-synuclein.

In one embodiment, the disease is a neurological disorder. In oneembodiment, the disease is selected from the group of neurologicaldisorders consisting of neuropathy, amyloidosis, cancer, an oculardisease or disorder, viral or microbial infection, inflammation,ischemia, neurodegenerative disease, seizure, behavioral disorders,lysosomal storage disease, Lewy body disease, post poliomyelitissyndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy,Parkinson's disease, multiple system atrophy, striatonigraldegeneration, tauopathies, Alzheimer disease, supranuclear palsy, priondisease, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakobsyndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wastingdisease, and fatal familial insomnia, bulbar palsy, motor neurondisease, nervous system heterodegenerative disorder, Canavan disease,Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander'sdisease, Tourette's syndrome, Menkes kinky hair syndrome, Cockaynesyndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome,hepatolenticular degeneration, Lesch-Nyhan syndrome, Unverricht-Lundborgsyndrome, dementia, Pick's disease, spinocerebellar ataxia, cancer ofthe CNS and/or brain, including brain metastases resulting from cancerelsewhere in the body. In one embodiment, the disease is selected fromthe group of neurological disorders consisting of Alzheimer's disease,Parkinson's disease, cancer of the CNS and/or brain, including brainmetastases resulting from cancer elsewhere in the body, and tauopathies.In one embodiment, the disease is selected from the group ofneurological disorders consisting of Alzheimer's disease, Parkinson'sdisease and tauopathies.

In one embodiment, the therapeutic antibody comprises an effectorfunction competent Fc-region. In one embodiment, the effector functioncompetent Fc-region is an Fc-region that specifically binds to/can bespecifically bound by human FcγR. In one embodiment, the effectorfunction competent Fc-region can elicit ADCC.

In one embodiment, ADCC elicited (upon injection/while binding to thesecond (cell surface) target) by the bispecific therapeutic antibody islower than that elicited by a bivalent bispecific antibody that has onlyone, i.e. exactly one, binding site that specifically bind to the first(cell surface) target and (exactly) one binding site that specificallybinds to the second (cell surface) target, i.e. one of the binding sitesspecifically binding to the first (cell surface) target is deleted. Inone embodiment, the ADCC is 10-fold or more lower.

In one embodiment, the administration is an intravenous, subcutaneous,or intramuscular administration.

In one embodiment, the first antibody heavy chain (of i)) and the secondantibody heavy chain (of ii)) form a heterodimer. In one embodiment, thefirst antibody heavy chain and the second antibody heavy chain comprisemutations supporting the formation of a heterodimer.

In one embodiment,

-   -   a) the antibody heavy chains are full-length antibody heavy        chains of the human subclass IgG1,    -   b) the antibody heavy chains are full-length antibody heavy        chains of the human subclass IgG4,    -   c) one of the antibody heavy chains is a full length antibody        heavy chain of the human subclass IgG1 with the mutations T366W        and optionally S354C or Y349C and the other antibody heavy chain        is a full length antibody heavy chain of the human subclass IgG1        with the mutations T366S, L368A, Y407V and optionally Y349C or        S354C,    -   d) both antibody heavy chains are full length antibody heavy        chains of the human subclass IgG1 with the mutations I253A,        H310A and H435A and the mutations T366W and optionally S354C or        Y349C in one of the antibody heavy chains and the mutations        T366S, L368A, Y407V and optionally Y349C or S3 54C in the        respective other antibody heavy chain,    -   e) both antibody heavy chains are full length antibody heavy        chains of the human subclass IgG1 with the mutations M252Y,        S254T and T256E and the mutations T366W and optionally S354C or        Y349C in one of the antibody heavy chains and the mutations        T366S, L368A, Y407V and optionally Y349C or S3 54C in the        respective other antibody heavy chain, or    -   f) both antibody heavy chains are antibody heavy chains of the        human subclass IgG1 with the mutations T307H and N434H and the        mutations T366W and optionally S354C or Y349C in one of the        antibody heavy chains and the mutations T366S, L368A, Y407V and        optionally Y349C or S354C in the respective other antibody heavy        chain.

In one embodiment,

-   -   a) the antibody heavy chains are antibody heavy chains of the        human subclass IgG1,    -   b) the antibody heavy chains are antibody heavy chains of the        human subclass IgG4,    -   c) one of the antibody heavy chains is an antibody heavy chain        of the human subclass IgG1 with the mutations T366W and        optionally S354C or Y349C and the other antibody heavy chain is        an antibody heavy chain of the human subclass IgG1 with the        mutations T366S, L368A, Y407V and optionally Y349C or S354C,    -   d) both antibody heavy chains are antibody heavy chains of the        human subclass IgG1 with the mutations I253A, H310A and H435A        and the mutations T366W and optionally S354C or Y349C in one of        the antibody heavy chains and the mutations T366S, L368A, Y407V        and optionally Y349C or S354C in the respective other antibody        heavy chain,    -   e) both antibody heavy chains are antibody heavy chains of the        human subclass IgG1 with the mutations M252Y, S254T and T256E        and the mutations T366W and optionally S354C or Y349C in one of        the antibody heavy chains and the mutations T366S, L368A, Y407V        and optionally Y349C or S354C in the respective other antibody        heavy chain, or    -   f) both antibody heavy chains are antibody heavy chains of the        human subclass IgG1 with the mutations T307H and N434H and the        mutations T366W and optionally S354C or Y349C in one of the        antibody heavy chains and the mutations T366S, L368A, Y407V and        optionally Y349C or S354C in the respective other antibody heavy        chain,    -   wherein the C-terminal lysine or glycine-lysine dipeptide is        present or absent.

EMBODIMENTS OF THE METHODS ACCORDING TO THE INVENTION

The relation between CSF, blood-brain-barrier and blood has beenreviewed by Katsinelos, T., et al. (Front. Immunol. 10 (2019) 1139) asfollows:

-   -   IgG levels are maintained in human serum at around 10 mg/ml. The        brain is isolated from serum by the blood-brain barrier (BBB),        which is impermeable to large macromolecules including IgG        (Neuwelt, E. A., et al., Nat. Rev. Neurosci. 12 (2011) 169-182).        The brain, instead, is bathed in cerebrospinal fluid (CSF),        which is produced following the filtration of blood and        transport of ions across the choroid plexus. The resulting        concentration of IgG in CSF is around 500- to 1,000-fold lower        than in serum. At face value, this low concentration of antibody        in the brain makes CNS antigens unattractive as targets for        passive immunotherapy, which is normally administered to the        periphery. This is compounded by a poor understanding of the        mechanisms by which steady state levels of antibody are        maintained. CSF flows around the brain, before exiting the CNS        along spinal and cranial nerves and via drainage to the        lymphatic system (Louveau, A., et al., Nature 523 (2015)        337-341; Aspelund, A., J. Exp. Med. 212 (2015) 991-999).        Intrathecally administered IgG is rapidly cleared from the        brain, largely through this bulk flow and with a possible        contribution of selective transport out of the brain. The        neonatal Fc receptor, FcRn, is expressed in abundance at the BBB        (Schlachetzki, F., et al., J. Neurochem. 81 (2002) 203-206).        Given FcRn's role in transcytosis of antibodies across the        placenta, it has been suggested that FcRn may perform reverse        transcytosis to help maintain the low IgG environment of the        CNS. There is some evidence that antibody clearance from the        brain is mediated in part by the antibody Fc domain (Zhang, Y.        and Pardridge, W. M., J. Neuroimmunol. 114 (2001) 168-172;        Cooper, P. R., et al., Brain Res. 1534 (2013) 13-21), and export        of an anti-Aβ monoclonal antibody was reduced in an        FcRn-deficient mouse (Deane, R., et al., J. Neurosci. 25 (2005)        11495-11503). However, the brain concentration of peripherally        administered IgG was not significantly different between        wild-type mice and mice lacking FcRn (Abuqayyas, L. and        Balthasar, J. P., Mol. Pharma. 10 (2013) 1505-1513).

For small experimental animals, the blood is removed from the brainprior to sampling by perfusion. For example, mice can be transcardiallyperfused with ice-cold PBS at a rate of 2 ml/min for 8 min. and brainsare subsequently harvested.

Methods to transport therapeutic antibodies across the blood brainbarrier, using multispecific antibodies, for example, bispecific ortrispecific antibodies, comprising one or more than one carriermolecule, and one or more than one cargo molecule, via areceptor-mediate transcytosis pathway are currently explored. Forexample, a transferrin receptor (TfR)-binding antibody (and variantsthereof) may be used as the carrier, and when fused to a cargo moleculeproduces a bispecific antibody that is able to cross the blood brainbarrier (see for example Zuchero, Y. J, Y., et. al., Neuron 89 (12016)70-82; Bien-Ly, N., et. al. J. Exp. Med. 211 (2014) 233-244; US2018/8002433; CA 3,000,560; which are incorporated herein by reference).Alternatively, an insulin-like growth factor 1 receptor (IGF-1R)-bindingantibody may be used as a carrier, and fused to the cargo molecule, toproduce a bispecific antibody that crosses the blood brain barrier (seefor example WO 2015/131256; WO 2015/131257; WO 2015/131258; which areincorporated herein by reference).

For a robust and correct determination of the amount in brain, lysatesof a therapeutic antibody transported across the blood-brain-barrierinto the brain the interference from residual blood in the sample has tobe excluded. As outlined above the resulting concentration of IgG in CSFis around 500- to 1,000-fold lower than in serum and the brain isspanned by an interwoven net of blood vessels. Thus, the chance ofresidual blood in brain tissue sample is not neglectable. Furthermore,even minor amounts of residual blood can severely interfere with thequantitative determination of antibody in brain tissue.

Thus, a correction, i.e. reduction, with the amount of therapeuticantibody in residual blood in the brain lysate sample has to be made.

Thus, the use of a quantitative blood correction marker that does notsignificantly diffuse during the perfusion phase into the brain isrequired. However, if these are determined at steady state, a small,constant concentration will exist behind the BBB.

The current invention is based, at least in part, on the finding thatthe amount of residual blood in a brain lysate can be determined byapplying a correction antibody shortly before the brain sample is taken.It has been found that it is especially advantageous to use as referenceantibody an antibody that is not specifically binding to any target inthe experimental animal from which the brain sample is obtained, mostpreferably a human germline antibody.

Thus, herein is reported a method for the determination of the amount ofa therapeutic antibody, which has been transported across theblood-brain-barrier from the blood into the brain of an experimentalanimal. The amount is preferably determined in a brain lysate sample.The gist of the invention lies in the additional application of an inertantibody, which is not transported across the blood-brain-barrier,shortly before obtaining the brain sample in which the amount of thetherapeutic antibody transported across the blood-brain-barrier has tobe determined. By applying the inert antibody, a correction value forthe amount of therapeutic antibody present in residual blood in thebrain sample is obtained. This residual blood-derived amount is used tocorrect the determined amount for non-brain-located antibody. Adetermination without correction would determine the total amount oftherapeutic antibody in the sample, i.e. the amount transported acrossthe blood-brain-barrier into the brain and the amount in residual bloodin the sample. The amount of therapeutic antibody in residual blood isnot neglectable, as only about 0.1% of the antibody in the blood willpass the blood-brain-barrier. Thus, the concentration of the therapeuticantibody in the blood exceeds the concentration of the therapeuticantibody in the brain by at least two and up to three orders ofmagnitude. Thereby the results obtained are too high if not correctedwith a method according to the current invention.

This is especially important for the comparator IgG or brain shuttleswith clearance approaching that of an IgG, because slowly clearingmolecules maintain a high concentration in the blood, and if the amountis comparatively very small in the brain, then a small amount of bloodcontamination can overwhelm the determination of brain concentrations.

The method according to the current invention can be applied to anybrain tissue sample independently of the method used for removing bloodtherefrom.

The method according to the invention has a cross-species assayavailability, sufficient assay-robustness, -precision and -accuracy anda broad sensitivity range.

In a nutshell, the current invention provides a method for determiningresidual blood in a brain sample of an experimental animal,

-   -   wherein just prior to perfusion, i.e. at most 5 min. before, a        second inert IgG is administered, a plasma sample is taken,        perfusion is conducted, brain and plasma concentrations are        measured and corrected    -   wherein the advantage is that the amount that transits the        blood-brain-barrier is limited; thereby, the concentration of        the second inert antibody reflects only plasma volume    -   wherein a first specific assay for the therapeutic antibody and        a second specific assay for the second inert antibody is        employed    -   wherein issues with anti-therapeutic antibody antibody positive        animals, which may confound the measurement, could be prevented.

FIG. 1 provides an exemplary calculation exploiting the use of an interantibody to determine blood contamination in a (brain) tissue sample.

For example, using a conventional ELISA the concentration of thetherapeutic monoclonal antibody (tmAb) and the inert referencemonoclonal antibody (refmAb) is determined in the blood plasma as wellas in the homogenized brain tissue sample. The result of an ELISA isnormally obtained as a mass concentration with the SI unit [g/L]. In afirst step each of the mass concentrations determined for the tmAb andthe refmAb, respectively, is converted into a mass fraction with theunit [g/g] by dividing the determined mass concentration by the braintissue concentration of the sample. In the second step the amount ofresidual plasma in the brain tissue sample, i.e. theplasma-contamination, is calculated by dividing the mass fraction of theinert antibody obtained in the first step by the determined plasmaconcentration of the refmAb. Thereby the volume of residual plasma perweight of brain sample is obtained. In a third step the mass fraction oftmAb in the brain tissue sample originating from plasma contamination iscalculated by multiplying the plasma concentration of the tmAb with thevolume of residual plasma per weight of brain sample. In the fourth andfinal step, the true brain concentration of the tmAb is obtained bysubtracting the mass fraction of tmAb in the brain tissue sampleoriginating from plasma contamination obtained in the third step fromthe mass fraction determined for the tmAb in the first step.

The method according to the invention has been applied to the analysisof two bispecific antibodies, binding to TfR and a therapeutic target 1or 2, respectively, in cynomolgus monkey brain lysates. The respectivestructure of the antibodies is shown in FIG. 2. The detection assays forthe therapeutic antibody and the reference antibody, respectively, areshown in FIG. 3.

As outlined in Example 1 the assay for the determination of the inertantibody has sensitivity of 8 ng/ml, i.e. about 1.1-1.5 μL bloodplasma/g cynomolgus brain can be detected (this corresponds to about2.2-3 μL blood/g cynomolgus brain).

Five different brain regions have been analyzed: Cerebellum,Hippocampus, Statium, Cortex and Choroid plexus.

Four different animals have been analyzed, whereof animal 1 to 3contained no residual blood in the brain samples but animal 4 did asdetermined by photo analysis (data not shown).

With the method according to the current invention, this contaminationcould be detected and, thus, the respective values could be correctedaccordingly.

DP47GS DP47GS PGLALA plasma blood PGLALA [ng/mL] concentrationcontamination [μg/mL] animal time Sample tissue MW [μL plasma/g brain][μL blood/g brain] in CPP 1 336 h Cerebellum BLQ 25 Hippocampus BLQStriatum BLQ Cortex BLQ Choroid plexus BLQ 2 336 h Cerebellum BLQ 23Hippocampus BLQ Striatum BLQ Cortex BLQ Choroid plexus BLQ 3 336 hCerebellum BLQ 33 Hippocampus BLQ Striatum BLQ Cortex BLQ Choroid plexusBLQ 4 336 h Cerebellum 18 1.6 3 27 Hippocampus BLQ Striatum  9 1.3 3Cortex 17 2.1 4 Choroid plexus BLQ

determined concentration plasma brain [ng/g] Choroid animal [ng/mL][ng/μL] Cerebellum Hippocampus Striatum Cortex plexus 1 antibody_1  350.034741 10.9  50.5  21.0  13.4  334    2 antibody_1 184 0.184316 21.5 47.5  28.7  21.8  744    3 antibody_1  20 0.019709 17.3  46.1  24.4 24.6  747    4 antibody_1 101 0.101429 17.5  45.1  28.2  25.1  385   brain tissue  0.422  0.164  0.262  0.295  0.019 concentration [g/mL]->corrected brain 17.3  27.9  24.9  concentration [ng/g]->

plasma brain [ng/mL] Choroid [ng/mL] [ng/μL] Cerebellum HippocampusStriatum Cortex plexus 1 inert 25107 25.107 BLQ BLQ BLQ BLQ BLQreference antibody 2 inert 25807 25.807 BLQ BLQ BLQ BLQ BLQ referenceantibody 3 inert 33193 33.193 BLQ BLQ BLQ BLQ BLQ reference antibody 4inert 27212 27.212 18 BLQ 9 17 BLQ reference antibody

In a further study fifteen animals have been does with an anti-Abetaantibody at 20 mg/kg and fifteen animals have been does with 10 mg/kg ofan anti-Abeta/TfR antibody. After different time points afteradministration, the respective samples have been analyzed. In all sampleresidual blood has been detected in the respective brain tissue samples.Thus, also in these cases corrected values were obtained with the methodaccording to the current invention.

antibody plasma time after ng/g μl/g analyte application animal Sampletissue tissue tissue Anti-Abeta  4 h 29211 Brain (Cerebellum) 22 1.0antibody Brain (Hippocampus) 21 0.9 Brain (Striatum) 20 0.9 Brain(Cortex) 26 1.2 Anti-Abeta  4 h 29213 Brain (Cerebellum) 16 0.6 antibodyBrain (Hippocampus) 20 0.7 Brain (Striatum) 13 0.5 Brain (Cortex) 15 0.6Brain (Choroid plexus) 822 29.6 Anti-Abeta  4 h 29214 Brain (Cerebellum)31 1.2 antibody Brain (Hippocampus) 38 1.4 Brain (Striatum) 21 0.8 Brain(Cortex) 25 1.0 Brain (Choroid plexus) 569 21.7 Anti-Abeta  24 h 29328Brain (Cerebellum) 53 3.7 antibody Brain (Hippocampus) 31 2.1 Brain(Striatum) 22 1.5 Brain (Cortex) 13 0.9 Brain (Choroid plexus) 925 63.8Anti-Abeta  24 h 29329 Brain (Cerebellum) 22 1.1 antibody Brain(Hippocampus) 22 1.0 Brain (Striatum) 21 1.0 Brain (Cortex) 23 1.1 Brain(Choroid plexus) 925 43.6 Anti-Abeta  24 h 29330 Brain (Cerebellum) 180.9 antibody Brain (Hippocampus) 25 1.3 Brain (Striatum) 25 1.2 Brain(Cortex) 28 1.4 Brain (Choroid plexus) 493 25.0 Anti-Abeta  96 h 29208Brain (Cerebellum) 29 1.1 antibody Brain (Hippocampus) 32 1.2 Brain(Striatum) 32 1.2 Brain (Cortex) 35 1.3 Brain (Choroid plexus) 411 15.0Anti-Abeta  96 h 29217 Brain (Cerebellum) 19 0.6 antibody Brain(Hippocampus) 42 1.3 Brain (Striatum) 22 0.7 Brain (Cortex) 23 0.7 Brain(Choroid plexus) 925 28.8 Anti-Abeta  96 h 29219 Brain (Cerebellum) 210.6 antibody Brain (Hippocampus) 21 0.6 Brain (Striatum) 16 0.5 Brain(Cortex) 28 0.8 Brain (Choroid plexus) 529 15.7 Anti-Abeta 168 h 29114Brain (Cerebellum) 33 1.1 antibody Brain (Hippocampus) 23 0.7 Brain(Striatum) 32 1.0 Brain (Cortex) 22 0.7 Brain (Choroid plexus) 493 15.9Anti-Abeta 168 h 29119 Brain (Cerebellum) 20 1.1 antibody Brain(Hippocampus) 19 1.0 Brain (Striatum) 26 1.4 Brain (Cortex) 28 1.5 Brain(Choroid plexus) 569 30.7 Anti-Abeta 168 h 29122 Brain (Cerebellum) 241.1 antibody Brain (Hippocampus) 18 0.8 Brain (Striatum) 26 1.2 Brain(Cortex) 21 1.0 Brain (Choroid plexus) 925 42.3 Anti-Abeta 336 h 29140Brain (Cerebellum) 34 1.5 antibody Brain (Hippocampus) 20 0.9 Brain(Striatum) 37 1.7 Brain (Cortex) 54 2.5 Brain (Choroid plexus) 569 25.7Anti-Abeta 336 h 29191 Brain (Cerebellum) 22 0.8 antibody Brain(Hippocampus) 23 0.9 Brain (Striatum) 21 0.8 Brain (Cortex) 22 0.8 Brain(Choroid plexus) 925 34.7 Anti-Abeta 336 h 29194 Brain (Cerebellum) 592.9 antibody Brain (Hippocampus) 22 1.1 Brain (Striatum) 16 0.8 Brain(Cortex) 33 1.6 Brain (Choroid plexus) 740 36.2 Anti-  4 h 29317 Brain(Cerebellum) 22 0.8 Abeta/TfR Brain (Hippocampus) 32 1.2 antibody Brain(Striatum) 20 0.8 Brain (Cortex) 23 0.9 Brain (Choroid plexus) 206 7.9Anti-  4 h 29319 Brain (Cerebellum) 13 0.6 Abeta/TfR Brain (Hippocampus)23 1.1 antibody Brain (Striatum) 28 1.3 Brain (Cortex) 13 0.6 Brain(Choroid plexus) 673 30.8 Anti-  4 h 29331 Brain (Cerebellum) 44 1.6Abeta/TfR Brain (Hippocampus) 22 0.8 antibody Brain (Striatum) 14 0.5Brain (Cortex) 19 0.7 Brain (Choroid plexus) 617 21.6 Anti-  24 h 29197Brain (Cerebellum) 32 1.2 Abeta/TfR Brain (Hippocampus) 23 0.9 antibodyBrain (Striatum) 69 2.7 Brain (Cortex) 22 0.8 Brain (Choroid plexus) 30811.9 Anti-  24 h 29199 Brain (Cerebellum) 21 0.7 Abeta/TfR Brain(Hippocampus) 20 0.6 antibody Brain (Striatum) 16 0.5 Brain (Cortex) 190.6 Brain (Choroid plexus) 296 9.6 Anti-  24 h 29201 Brain (Cerebellum)21 0.6 Abeta/TfR Brain (Hippocampus) 19 0.6 antibody Brain (Striatum) 180.6 Brain (Cortex) 17 0.5 Brain (Choroid plexus) 389 11.7 Anti-  96 h29207 Brain (Cerebellum) 16 0.6 Abeta/TfR Brain (Hippocampus) 21 0.8antibody Brain (Striatum) 25 0.9 Brain (Cortex) 30 1.1 Brain (Choroidplexus) 389 14.3 Anti-  96 h 29218 Brain (Cerebellum) 26 1.0 Abeta/TfRBrain (Hippocampus) 24 0.9 antibody Brain (Striatum) 16 0.6 Brain(Cortex) 23 0.9 Brain (Choroid plexus) 822 32.1 Anti-  96 h 29220 Brain(Cerebellum) 17 0.7 Abeta/TfR Brain (Hippocampus) 15 0.6 antibody Brain(Striatum) 21 0.9 Brain (Cortex) 17 0.7 Brain (Choroid plexus) 389 16.3Anti- 168 h 29118 Brain (Cerebellum) 17 0.6 Abeta/TfR Brain(Hippocampus) 19 0.7 antibody Brain (Striatum) 19 0.7 Brain (Cortex) 200.7 Brain (Choroid plexus) 493 18.4 Anti- 168 h 29141 Brain (Cerebellum)21 1.0 Abeta/TfR Brain (Hippocampus) 32 1.6 antibody Brain (Striatum) 452.2 Brain (Cortex) 20 1.0 Brain (Choroid plexus) 1057 51.9 Anti- 168 h29157 Brain (Cerebellum) 24 0.7 Abeta/TfR Brain (Hippocampus) 24 0.6antibody Brain (Striatum) 50 1.4 Brain (Cortex) 18 0.5 Brain (Choroidplexus) 1057 28.8 Anti- 240 h 28640 Brain (Cerebellum) 29 1.4 Abeta/TfRBrain (Hippocampus) 22 1.0 antibody Brain (Striatum) 20 0.9 Brain(Cortex) 19 0.9 Brain (Choroid plexus) 1057 49.9 Anti- 240 h 28641 Brain(Cerebellum) 14 0.4 Abeta/TfR Brain (Hippocampus) 16 0.5 antibody Brain(Striatum) 26 0.8 Brain (Cortex) 19 0.6 Brain (Choroid plexus) 463 14.5Anti- 240 h 29139 Brain (Cerebellum) 21 0.9 Abeta/TfR Brain(Hippocampus) 17 0.7 antibody Brain (Striatum) 22 0.9 Brain (Cortex) 120.5 Brain (Choroid plexus) 218 9.4

To show the general applicability of the method according to the currentinvention the same analysis has been done with a second antibody, ananti-TfR/target 2 antibody, in C57BL/6 wild-type mice.

An overlay of the calibration curves of the detection assay of the inertreference antibody in the presence of 1% cynomolgus brain lysate (CBL;cynoBL) and 1% mouse brain lysate (MBL; muBL) is shown in FIG. 4. It canbe seen that the origin of the matrix does not influence the assay.

The working range for the assay in the presence of 1% MBL is 8.4 ng/mLto 250 ng/mL. Up to 10 μg/mL of therapeutic antibody can be present inthe assay without interference in the presence of 1 MBL.

The working range for the assay in the presence of 1% mouse pooledplasma (MPP) is 11 ng/mL to 220 ng/mL. Up to 20 μg/mL of therapeuticantibody can be present in the assay without interference in thepresence of 1% MPP.

A single dose of 20 mg/ml of the antibody was applied and sample wereanalyzed 24 h, 48 h, 96 h, 168 h, 336 h, 504 h and 672 h afterapplication. The respective concentrations in brain lysate and plasmahave been determined. In FIG. 5, the determined concentrations of theapplied antibody in brain lysate are shown as ratio of uncorrected vs.corrected brain concentration. That is, if the correction does notinfluence the value then the ratio is 1. If due to the correction by theresidual plasma value the determined concentration of the secondantibody has been reduced the value will become smaller than 1. Thisdifference increases by time as more antibody is transported across theblood-brain-barrier. It can be seen from FIG. 5 that the ratio becomessmaller and smaller by time. Thereby it can be seen that the correctiondone according to the method according to the current inventioneliminated interference from residual blood in the brain samples. Therespective assay used for the determination of the second antibody isshown in FIG. 6.

Inert Reference Monoclonal Antibody of the Method According to theInvention

An inert reference monoclonal antibody useful in the method according tothe current invention is preferably a human immunoglobulin molecule,especially a human immunoglobulin molecule that is not capable ofspecific binding to an antigen.

An exemplary inert reference monoclonal antibody is the antibody DP47GS.DP47GS comprises a heavy chain variable region sequence based on thehuman VH3-23 germline sequence and a light chain variable regionsequence based on the human Vk3-20 germline sequence.

In one embodiment, said inert reference monoclonal antibody is anIgG-class immunoglobulin molecule, particularly an IgG1-subclassimmunoglobulin molecule. In one embodiment, said inert referencemonoclonal antibody is a human immunoglobulin molecule. In oneembodiment, said inert reference monoclonal antibody is a monoclonalantibody. In one embodiment, said inert reference monoclonal antibody isnot capable of specific binding to an antigen. In one embodiment, saidinert reference monoclonal antibody comprises a heavy chain variableregion sequence based on the human VH3-23 germline sequence. In aspecific embodiment, said inert reference monoclonal antibody comprisesthe heavy chain variable region sequence of SEQ ID NO: 67. In oneembodiment, said inert reference monoclonal antibody comprises a lightchain variable region sequence based on the human Vk3-20 germlinesequence. In a specific embodiment, said inert reference monoclonalantibody comprises the light chain variable region sequence of SEQ IDNO: 68. In an even more specific embodiment, said inert referencemonoclonal antibody comprises the heavy chain variable region sequenceof SEQ ID NO: 67 and the light chain variable region sequence of SEQ IDNO: 68. In one embodiment, said inert reference monoclonal antibody isnot capable of specific binding to an antigen, and comprises a heavychain variable region sequence based on the human VH3-23 germlinesequence and a light chain variable region sequence based on the humanVk3-20 germline sequence.

In one embodiment, said inert reference monoclonal antibody comprises aheavy chain variable region sequence based on the human VH3-23 germlinesequence. In a specific embodiment, said inert reference monoclonalantibody comprises a heavy chain variable region sequence that is atleast 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQID NO: 67. In one embodiment, said inert reference monoclonal antibodycomprises a light chain variable region sequence based on the humanVk3-20 germline sequence. In a specific embodiment, said inert referencemonoclonal antibody comprises a light chain variable region sequencethat is at least 95%, 96%, 97%, 98%, 99% or 100% identical to thesequence of SEQ ID NO: 68. In an even more specific embodiment, saidinert reference monoclonal antibody comprises the heavy chain variableregion sequence of SEQ ID NO: 67 and the light chain variable regionsequence of SEQ ID NO: 68. Immunoglobulin molecules comprising thesevariable region sequences are not capable of specific binding to anantigen, particularly a human antigen. They lack binding to normaltissues as well as PBMCs, have no polyreactivity and show nonon-specific accumulation in vivo by imaging (data not shown). Thevariable region sequences are entirely based on human germlinesequences, with the exception of the heavy chain CDR 3 wherein a GSGsequence has been introduced to generate a non-binding immunoglobulin.

In one embodiment, said inert reference monoclonal antibody comprises aheavy chain with a variable domain with the amino acid sequence of SEQID NO: 67 and a human IgG1 constant region and a light chain with avariable domain with the amino acid sequence of SEQ ID NO: 68 and ahuman kappa light chain constant domain. In one embodiment, said inertreference monoclonal antibody comprises in the heavy chain Fc-region themutations L234A, L235A and P329G (numbering according to Kabat EUindex).

In one embodiment, said inert reference monoclonal antibody comprises aheavy chain with the amino acid sequence of SEQ ID NO: 69 and a lightchain with the amino acid sequence of SEQ ID NO: 70.

Comparative Methods and Results Comparative Technical Approach:

Correction by Residual Blood Volume without Perfusion

Friden et al. (J. Cerebral Blood Flow & Met 30 (2010) 150-161) collectedthe available information from literature on brain vascular space (cf.Table 1 of Friden et al.).

Based on the most commonly used 14C-Dextran method, brain plasma valuewould amount for approx. 18.1 μL/g brain tissue. When applying thiscorrection all determined values became negative.

Thus, simply assuming that the total brain plasma value would be presentin a brain tissue sample was not correct.

Thus, no absolute value could be applied but a co-determined correctionfactor was required.

Thus, a different correction factor was required.

As perfusion will be performed, a control for remaining bloodcontamination is required. This is especially important for thecomparator IgG or brain shuttles with clearance approaching that of anIgG—why? Because slowly clearing molecules maintain a high concentrationin the blood, and if the amount is comparatively very small in thebrain, then a small amount of blood contamination can overwhelm thedetermination of brain concentrations.

Thus, there is the need to use a quantitative blood correction markerthat does not significantly diffuse during the application as well asthe perfusion phase in to the brain.

Comparative Markers:

Different other non-antibody inert reference molecules, which were,prior to their testing, deemed to be likewise suitable as a correctivemeans in the method according to the current invention are otherendogenous proteins with high molecular weight and high endogenous bloodlevels.

Determination of Complement Factor H

Different publications indicated that in cynomolgus cerebrospinal fluid(liquor cerebrospinalis) as well as cynomolgus brain lysates nocomplement factor H is present, wherein it is common in non-CSF ornon-brain tissue. Therefore, it has been assumed that the detection ofcomplement factor H is a viable surrogate marker for the determinationof residual contaminating blood in cCSF and CBL samples.

As positive controls human pooled serum (HPS; 200-800 μg/mL complementfactor H) and human pooled plasma (HPP; about 300 μg/mL complementfactor H) were available.

The assay was set-up as an Elecsys-assay (Roche Diagnostics GmbH,Mannheim, Germany). The respective calibration curve is shown in FIG. 7.The working range of this assay was between 7.8 μg/mL and 2000 μg/mL.

sample μg/mL positive control human pooled plasma 322 human pooled serum324  1 cynomolgus pooled serum 1.4  2 cynomolgus pooled plasma 1 1.4  3cynomolgus pooled plasma 2 1.4  4 animal 01 HH8061813 1.2  5 animal 02HH8061813 1.2  6 animal 12 HH7061813 1.2  7 animal 07 HH7061813 1.0  8animal 08 HH7061813 1.0  9 cynomolgus pooled plasma 1 1.1 10 cynomolguspooled plasma 2 1.3 11 (replica of 1) cynomolgus pooled serum 1.5negative control cynomolgus brain lysate animal 1 blank value cynomolgusbrain lysate animal 2 blank value cynomolgus cerebrospinal fluid blankvalue

Thus, it has been found that the determination of complement factor H isnot suited as surrogate marker for residual contaminating blood as theassay is not sensitive enough.

Determination of Alpha-2-Macroglobulin

Different publications indicated that in cynomolgus cerebrospinal fluid(liquor cerebrospinalis) as well as cynomolgus brain lysates noα2-macroglobulin is present, wherein it is common in non-CSF ornon-brain tissue (1500-2000 μg/mL). Therefore, it has been assumed thatthe detection of α2-macroglobulin is a viable surrogate marker for thedetermination of residual contaminating blood in cCSF and CBL samples.

The assay principle of the ELISA-assay for the determinationα2-macroglobulin is shown in FIG. 8 and a respective calibration curvein FIG. 9.

The assay had a working range from 0.62 ng/mL (LLOQ) to 39 ng/mL (ULOQ).

human pooled serum 2.8 g/l cynomolgus pooled serum 1  36 ng/mlcynomolgus pooled serum 2  44 ng/ml cynomolgus brain lysate sample   0ng/ml

The expected values for human serum and plasma could be confirmedwhereas in cynomolgus pooled serum only 1/25,000 of the expected amountcould be detected. Thus, this value is too low to be quantified indiluted form in cynomolgus cerebrospinal fluid and brain lysates. Thus,the determination of α2-macroglobulin is not suited as surrogate marker.

Determination of Complement Component 5a (C5a)

Different publications indicated that in cynomolgus cerebrospinal fluid(liquor cerebrospinalis) as well as cynomolgus brain lysates nocomplement component 5a is present, wherein it is common in non-CSF ornon-brain tissue (60-110 μg/mL in human serum). Therefore, it has beenassumed that the detection of complement component C5a is a viablesurrogate marker for the determination of residual contaminating bloodin cCSF and CBL samples.

Like for α2-macroglobulin an ELISA has been set-up with a murineanti-human C5a antibody as capture antibody and a biotinylated murineanti-human C5a antibody as detection antibody, whereby both antibodiesbind to non-interfering epitopes on human C5a. A respective calibrationcurve is shown in FIG. 10.

The assay had a working range from 0.03 ng/mL (LLOQ) to 2 ng/mL (ULOQ).

final concentration in matrix human pooled serum   57 μg/mL human pooledplasma 24.5 μg/mL cynomolgus pooled serum 1   52 μg/mL cynomolgus pooledserum 2   70 μg/mL cynomolgus brain lysate   11 μg/mL

Thus, it has been found that C5a can be determined in CBL samples.

Thus, the determination of C5a is not suited as surrogate marker.

Use of Magnevist®

Magnevist® (gadopentetat-dimeglumin) is a MRT-imaging agent. It wasassumed that Magnevist® will not passage the blood-brain-barrier.

A pharmacokinetic study showed that up to 15 minutes only the measuredbrain concentration represents the blood compartment correctly. Afterthis time, there is diffusion of Magnevist® into the brain tissue,confounding the applied correction. The respective time-course is shownin FIG. 11. At the 5-minute time point, plasma volume is estimated as14.1 μL/g brain.

Thus, this approach cannot be sued with perfusion, as the time taken forperfusion would lead to a diffusion of Magnevist across theblood-brain-barrier—confounding the residual blood correction.

Determination of Cynomolgus IgG in Cynomolgus Cerebrospinal Fluid (cCSF)

Different publications indicated that in cynomolgus cerebrospinal fluid(liquor cerebrospinalis) only minor amounts of cynomolgus IgG arepresent. Therefore, it has been assumed that the detection of total Igin cCSF is a viable surrogate marker for the direct determination oftransported therapeutic antibody. Therefore, a bridging ELISA was set upas shown in FIG. 12. To exclude matrix effects a human IgG-depleted cCSFwas generated by incubating the cCSF with anti-human CH1/kappa antibodybound to magnetic beads.

The respective calibration curves with buffer and human IgG-depletedcCSF are shown in FIG. 13. It can be seen that no matrix effect occurs.

The assay had a working range of from 120 ng/ml to 7.2 ng/mL IgG.

Using this assay it has been found that in cynomolgus pooled plasmasamples (CPP) about 11-19 mg/mL IgG could be detected, whereas in cCSFsamples substantial amounts of about 4-18 μg/mL cynomolgus IgG could bedetected.

The following examples, sequences and figures are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

Description of the Sequences

SEQ ID NO description 01 human Aβ42 02 (G4S)4 linker 03 (G4S)6G2 linker04 mAb 0012 05 mAb 0012 06 mAb 0012 07 mAb 0012 08 mAb 0015 09 mAb 001510 mAb 0015 11 mAb 0015 12 mAb 0020 13 mAb 0020 14 mAb 0020 15 mAb 002416 mAb 0024 17 mAb 0024 18 mAb 0024 19 A-beta binding site VH 20 A-betabinding site VL 21 transferrin receptor binding site VH 22 transferrinreceptor binding site VL 23 light chain 24 heavy chain 25 light chain 26heavy chain Fab fragment 27 anti-transferrin receptor antibody 128.1 VH28 anti-transferrin receptor antibody 128.1 VL 29 heavy chain variabledomain 30 light chain variable domain 31 anti-transferrin receptorbinding site VH 32 anti-transferrin receptor binding site VL 33 theanti-transferrin receptor binding site HVR-H1 34 the anti-transferrinreceptor binding site HVR-H2 35 the anti-transferrin receptor bindingsite HVR-H3 36 the anti-transferrin receptor binding site HVR-H3 37 theanti-transferrin receptor binding site HVR-H3 38 the anti-transferrinreceptor binding site HVR-L1 39 the anti-transferrin receptor bindingsite HVR-L2 40 the anti-transferrin receptor binding site HVR-L3 41heavy chain variable domain anti-CD20 antibody 42 light chain variabledomain anti-CD20 antibody 43 heavy chain variable domain anti-alphasynuclein (asyn) antibody 44 light chain variable domain anti-alphasynuclein antibody 45 heavy chain variable domain anti-alpha synucleinantibody 46 light chain variable domain anti-alpha synuclein antibody 47heavy chain variable domain anti-alpha synuclein antibody 48 light chainvariable domain anti-alpha synuclein antibody 49 heavy chain variabledomain anti-alpha synuclein antibody 50 light chain variable domainanti-alpha synuclein antibody 51 heavy chain variable domain anti-alphasynuclein antibody 52 light chain variable domain anti-alpha synucleinantibody 53 heavy chain variable domain anti-alpha synuclein antibody 54light chain variable domain anti-alpha synuclein antibody 55glucocerebrosidase 56 peptidic linker 57 peptidic linker 58 brain target59 brain target 60 brain target 61 brain target 62 the anti-transferrinreceptor binding site HVR-H1 63 the anti-transferrin receptor bindingsite HVR-H2 64 the anti-transferrin receptor binding site HVR-H3 65 theanti-transferrin receptor binding site HVR-L1 66 the anti-transferrinreceptor binding site HVR-L3 67 heavy chain variable region sequenceDP47GS 68 light chain variable region sequence DP47GS 69 inert referencemonoclonal antibody comprises a heavy chain 70 inert referencemonoclonal antibody comprises a light chain

DESCRIPTION OF THE FIGURES

FIG. 1 Exemplary calculation for determining residual-plasma-correctedbrain lysate concentration of a tmAb.

FIG. 2 Structure of the exemplary brain-shuttle construct used in theexamples of the method according to the invention.

FIG. 3 Detection assay for therapeutic antibody and reference antibodyused in the Examples.

FIG. 4 Overlay of the calibration curves of the detection assayaccording to Example 1 for the inert reference antibody in the presenceof 1% cynomolgus and mouse, respectively, brain lysate.

FIG. 5 Ratio of corrected concentration to non-corrected concentrationof antibody 2 in mouse brain lysate.

FIG. 6 ELISA for the determination of antibody 2.

FIG. 7 Calibration curve of complement factor H Elecsys assay.

FIG. 8 ELISA for the quantification of α2-macroglobulin in cCSF. Thecapture antibody is a murine anti-human α2-macroglobulin antibody; thedetection antibody is a biotinylated goat anti-human α2-macroglobulinantibody.

FIG. 9 Calibration curve of the ELISA of Figure Z.

FIG. 10 Calibration curve of complement component 5a ELISA assay.

FIG. 11 Time course of Magnevist in PK study in plasma and brain tissue.

FIG. 12 ELISA for the quantification of cynomolgus IgG in cCSF. Thecapture mAb is an anti-cyno IgG antibody; the detection mAb is ananti-cyno IgG antibody binding to a not interference epitope withrespect to the first antibody.

FIG. 13 Calibration curve of the ELISA of Figure X.

General Methods Preparation of Cynomolgus Brain Tissue Homogenates

Frozen cynomolgus/mouse brain tissue samples of 300 mg were thawed atroom temperature for 2 h. 800 μL of lysis buffer, one tablet of cOmpleteprotease inhibitor cocktail (Roche Diagnostics GmbH) dissolved in 50 mLTissue Extraction Reagent I (Invitrogen), were added to the thawed braintissue. Next, the sample was homogenized in a MagNA Lyser instrument(Roche Diagnostics) for 20 seconds at 6500 rpm. The tissue homogenatewas then centrifuged for 10 minutes at 12,000 rpm using a Centrifuge5430 (Eppendorf). Finally, the supernatant was transferred to a 1.5 mLvial for further analysis or stored at −80° C.

Example 1 ELISA for the Quantification of DP47GS-PGLALA in Brain Lysates

To quantify the inert reference monoclonal antibody DP47GS-PGLALA (SEQID NO: 69 and 70) in cynomolgus brain lysate samples a serial sandwichenzyme linked immunosorbent assay (ELISA) was used. In the ELISAprocedure, all samples and controls are subjected to an initial 1:100pre-dilution in Assay Diluent to the desired 1% final assayconcentration.

Capture Antibody (anti-DP47GS antibody, biotinylated), dilutedCalibrators (DP47GS-PGLALA) as well as diluted Quality controls andsamples, detection reagent (anti-PGLALA antibody clone M-1.7.24,digoxygenylated) and anti-digoxygenin-antibody-POD-conjugate are addedsuccessively to a streptavidin coated microtiter plate (SA-MTP). Thereagents were incubated for 1 hour on a MTP shaker at 500 rpm and aftereach step the MTP was washed three times with 300 washing buffer (1×PBS,0.05% Tween) and residual fluids were removed. After that, the formedimmobilized immune complexes were visualized by addition of ABTSsolution, a horseradish POD substrate, which was converted to a coloredreaction product. Finally, the color intensity was photometricallydetermined (absorption at 405 nm-490 nm reference wavelength). Thesignal is proportional to the analyte concentration in the brain lysatesample. The quantification of DP47GS-PGLALA was performed by backcalculation of the absorbance values using the corresponding calibrationcurve with a non-linear 4-parameter Wiemer-Rodbard curve fittingfunction with weighting.

brain lysate assay concentration concentration DP47GS- DP47GS- PGLALAPGLALA Quality control Calculation [ng/mL] [ng/mL] ULQC highestcalibrator 250 2.5 Upper limit of quantification HQC highest calibrator× 185 1.85 High range 0.75 MQC geometric mean of 80 0.80 Medium rangeHQC and LQC LQC LLQC × 3 25 0.25 Low range LLQC lowest calibrator 8 0.08Lower limit of quantification

Coating with capture reagent is achieved by pipetting 100 μL of asolution comprising 500 ng/mL biotinylated anti-DP47GS antibody intoeach SA-MTP well. Thereafter the MTP is covered with adhesive cover foiland incubated for 1 hour on a MTP shaker (500 rpm). The supernatant isremoved and each well of the MTP is washed three times with 300 μLwashing buffer (PBS, 0.05% Tween). Residual washing buffer is carefullyremoved.

Then 100 μl of the respective calibrators, quality controls and samplesto the designated wells of the coated MTP. Thereafter the MTP is coveredwith adhesive cover foil and incubated for 1 hour on a MTP shaker (500rpm). The supernatant is removed and each well of the MTP is washedthree times with 300 μL washing buffer (PBS, 0.05 Tween). Residualwashing buffer is carefully removed.

Then 100 μL of digoxygenylated anti-PGLALA antibody clone M-1.7.24 at aconcentration of 125 ng/mL is added to each MTP well. Thereafter the MTPis covered with adhesive cover foil and incubated for 1 hour on a MTPshaker (500 rpm). The supernatant is removed and each well of the MTP iswashed three times with 300 μL washing buffer (PBS, 0.05 Tween).Residual washing buffer is carefully removed.

The 100 μL of an anti-digoxygenin antibody-POD-conjugate at aconcentration of 50 mU/mL is added to each MTP well. Thereafter the MTPis covered with adhesive cover foil and incubated for 1 hour on a MTPshaker (500 rpm). The supernatant is removed and each well of the MTP iswashed three times with 300 μL washing buffer (PBS, 0.05 Tween).Residual washing buffer is carefully removed.

Then 100 μL ABTS solution is added to each MTP well. The optical densityis measured until the average signal of the duplicates of Calibratorsample 1 reaches 1.8-2.2 AU at a measuring wavelength of 405 nm(reference wavelength 490 nm).

Example 2 ELISA for the Quantification of Cynomolgus IgG in CSF

To quantify cynomolgus IgG in cynomolgus cerebrospinal fluid a serialsandwich enzyme linked immunosorbent assay (ELISA) was used. In theELISA procedure, all samples and controls are subjected to an initialpre-dilution in Assay Diluent to the desired 1% final assayconcentration.

Capture Antibody (anti-cynomolgus IgG antibody 1; epitope 1;biotinylated), diluted Calibrators as well as diluted Quality controlsand samples, detection reagent (anti-cynomolgus IgG antibody 2; epitope2, not interfering with epitope 1; digoxygenylated) andanti-digoxygenin-antibody-POD-conjugate are added successively to astreptavidin coated microtiter plate (SA-MTP). The reagents wereincubated for 1 hour on a MTP shaker at 500 rpm and after each step theMTP was washed three times with 300 μL washing buffer (1×PBS, 0.05%Tween) and residual fluids were removed. After that, the formedimmobilized immune complexes were visualized by addition of ABTSsolution, a horseradish POD substrate, which was converted to a coloredreaction product. Finally, the color intensity was photometricallydetermined (absorption at 405 nm-490 nm reference wavelength). Thesignal is proportional to the analyte concentration in the brain lysatesample. The quantification of cynomolgus IgG was performed by backcalculation of the absorbance values using the corresponding calibrationcurve with a non-linear 4-parameter Wiemer-Rodbard curve fittingfunction with weighting.

cCSF assay concentration concentration cynomolgus cynomolgus Qualitycontrol Calculation IgG [ng/mL] IgG [ng/mL] ULQC highest calibrator12000 120 Upper limit of quantification HQC highest calibrator × 9000 90High range 0.75 MQC geometric mean 45000 45 Medium range of HQC and LQCLQC LLQC × 3 2200 22 Low range LLQC lowest calibrator 720 7.2 Lowerlimit of quantification

Coating with capture reagent is achieved by pipetting 100 μL of asolution comprising 250 ng/mL biotinylated anti-cynomolgus IgG antibody1 into each SA-MTP well. Thereafter the MTP is covered with adhesivecover foil and incubated for 1 hour on a MTP shaker (500 rpm). Thesupernatant is removed and each well of the MTP is washed three timeswith 300 μL washing buffer (PBS, 0.05 Tween). Residual washing buffer iscarefully removed.

Then 100 μl of the respective calibrators, quality controls and samplesto the designated wells of the coated MTP Thereafter the MTP is coveredwith adhesive cover foil and incubated for 1 hour on a MTP shaker (500rpm). The supernatant is removed and each well of the MTP is washedthree times with 300 μL washing buffer (PBS, 0.05 Tween). Residualwashing buffer is carefully removed.

Then 100 μL of digoxygenylated anti-cynomolgus antibody 2 at aconcentration of 250 ng/mL is added to each MTP well. Thereafter the MTPis covered with adhesive cover foil and incubated for 1 hour on a MTPshaker (500 rpm). The supernatant is removed and each well of the MTP iswashed three times with 300 μL washing buffer (PBS, 0.05 Tween).Residual washing buffer is carefully removed.

The 100 μL of an anti-digoxygenin antibody-POD-conjugate at aconcentration of 25 mU/mL is added to each MTP well. Thereafter the MTPis covered with adhesive cover foil and incubated for 1 hour on a MTPshaker (500 rpm). The supernatant is removed and each well of the MTP iswashed three times with 300 μL washing buffer (PBS, 0.05 Tween).Residual washing buffer is carefully removed.

Then 100 μL ABTS solution is added to each MTP well. The optical densityis measured until the average signal of the duplicates of Calibratorsample 1 reaches 1.8-2.2 AU at a measuring wavelength of 405 nm(reference wavelength 490 nm).

Example 3 Production of Brain Tissue Lysates

First, the lysis buffer was freshly prepared according to themanufacturer's instructions (Invitrogen; tissue extraction reagent I;Cat.-No. FNN0071). Per 50 ml of lysis buffer 1 tables of Complete isadded (Roche Diagnostics GmbH, Mannheim, Germany; Cat.-No. 11697498001).

Second, to the brain tissue sample, approx. 100-300 mg, between 600 μLand 800 were lysis buffer is added. Optionally MagNA Lyser Green Beadsare added.

Third, the samples were placed for 20 sec. at 6500 rpm in the MagNALyser (Roche Diagnostics GmbH, Mannheim, Germany).

Fourth, after incubation in the MagNA Lyser the samples are centrifugedfor 10 min. at 12,000 rpm (Eppendorf Centrifuge 5430).

Fifth, the supernatant (500-700 μL) was recovered and stored at −80° C.until further analysis.

1. A method or assay for determining the concentration of a therapeuticantibody in a tissue of an experimental animal, whereby the tissue has abarrier to the blood circulation of said animal and whereby thetherapeutic antibody had been administered to said experimental animal,wherein the interference from residual blood in a tissue sample of theexperimental animal, which is used for determining the concentration ofthe therapeutic antibody in said tissue, is reduced, the methodcomprising the following steps i) determining the concentration of thetherapeutic antibody in a blood sample of the experimental animal, ii)determining the concentration of the therapeutic antibody in the tissuesample of the experimental animal, iii) determining the concentration ofan inert reference antibody in the blood sample of the experimentalanimal, iv) determining the concentration of the inert referenceantibody in the tissue sample of the experimental animal, v) determiningthe tissue concentration in the tissue sample, and determining theconcentration of the therapeutic antibody in the tissue of theexperimental animal with the following formula:$\left. {\left. {\left. {\left. {\left. {{C_{{tmAb},{tissue}} = {\frac{C_{{tmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}} - {\frac{\frac{C_{{refmAb},{tissue},{\det.}}}{C_{{tis{sue}},{sample}}}}{C_{{refmAb},{{plasma}.\det.}}}*{C_{{tmAb},{plasma},\det}.{with}}}}}{C_{{tmAb},{plasma},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}i}}} \right){C_{{tmAb},{tissue},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{ii}}}} \right){C_{{refmAb},{tissue},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{iii}}}} \right){C_{{refmAb},{plasma},{\det.}} = {{concentration}{of}{the}{therapeutic}{antibody}{of}{iv}}}} \right){C_{{tissue},{sample}} = {{tissue}{concentration}{of}v}}} \right)$whereby the inert reference antibody does not cross said barrier betweenthe tissue and the blood circulation, whereby the inert referenceantibody had been administered i) either together with the therapeuticantibody in case the sample is to be taken within 5 minutes after theadministration of the therapeutic antibody, or ii) 2 to 10 minutes priorto taking the tissue sample, whereby the blood sample is taken directlyprior to the tissue sample.
 2. The Method according to claim 1, whereinthe tissue is either brain tissue and the therapeutic antibody can crossthe blood-brain-barrier or ocular tissue and the therapeutic antibodycan cross the blood-ocular-barrier.
 3. The method according to any oneof claims 1 to 2, wherein the therapeutic antibody is a bispecificantibody.
 4. The method according to any one of claims 1 to 3, whereinthe therapeutic antibody is specifically binding to human transferrinreceptor and a brain target.
 5. The method according to claim 4, whereinthe brain target is human CD20 or human Abeta or human alpha-synucleinor human tau or human glucocerebrosidase or human lingo-1 or humanhuntingtin.
 6. The method according to any one of claims 1 to 5, whereinthe experimental animal is selected from mouse, rat, rabbit, dog, sheep,ape, and monkey.
 7. The method according to any one of claims 1 to 6,wherein the experimental animal is a non-human experimental animal witha body weight of more than 100 g and less than 15 kg.
 8. The methodaccording to any one of claims 1 to 7, wherein the experimental animalis a cynomolgus monkey.
 9. The method according to any one of claims 1to 8, wherein the inert reference antibody is a human germline antibody.10. The method according to any one of claims 1 to 9, wherein the inertreference antibody is DP47GS.
 11. The method according to any one ofclaims 1 to 10, wherein the inert reference antibody does not cross saidbarrier in detectable amounts within 15 minutes after its application.12. The method according to claim 11, wherein the inert referenceantibody does not cross said barrier in detectable amounts within 10minutes after its application.
 13. The method according to any one ofclaims 1 to 12, wherein the inert antibody is administered about 5minutes prior to taking the tissue sample.
 14. The method according toany one of claims 1 to 10, wherein the tissue is perfused with anaqueous solution directly after taking the blood sample and prior totaking the tissue sample.
 15. The method according to any one of claims1 to 11, wherein the determining of the concentrations is by a bridgingELISA.